Structured Review

Millipore pet28a
Purification of recombinant BurI protein from insoluble fraction of induced E. coli BL21 harboring <t>pET28a-</t> burI . Lane M, protein marker in kDa; Lane 1, precipitation dissolved in 8M urea; lane 2, flow through; lane 3, resin after elution step; lane 4, wash fraction using 8M urea, lane 5, wash fraction using 8M urea containing 20 mM imidazole; lane 6, eluted fraction using 8M urea containing 500 mM imidazole. The recombinant BurI protein was successfully purified from its inclusion bodies with fairly good purity.
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Images

1) Product Images from "Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4"

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

Journal: PeerJ

doi: 10.7717/peerj.1117

Purification of recombinant BurI protein from insoluble fraction of induced E. coli BL21 harboring pET28a- burI . Lane M, protein marker in kDa; Lane 1, precipitation dissolved in 8M urea; lane 2, flow through; lane 3, resin after elution step; lane 4, wash fraction using 8M urea, lane 5, wash fraction using 8M urea containing 20 mM imidazole; lane 6, eluted fraction using 8M urea containing 500 mM imidazole. The recombinant BurI protein was successfully purified from its inclusion bodies with fairly good purity.
Figure Legend Snippet: Purification of recombinant BurI protein from insoluble fraction of induced E. coli BL21 harboring pET28a- burI . Lane M, protein marker in kDa; Lane 1, precipitation dissolved in 8M urea; lane 2, flow through; lane 3, resin after elution step; lane 4, wash fraction using 8M urea, lane 5, wash fraction using 8M urea containing 20 mM imidazole; lane 6, eluted fraction using 8M urea containing 500 mM imidazole. The recombinant BurI protein was successfully purified from its inclusion bodies with fairly good purity.

Techniques Used: Purification, Recombinant, Marker, Flow Cytometry

SDS-PAGE profile of overproduction of BurI in E. coli BL21 (DE3) pLysS followed by CBB staining. SDS-PAGE analysis on (A) cell lysate and (B) on soluble and insoluble fraction of cell lysate after centrifugation at 14,000 rpm. Cell lysate of E. coli harboring pET28a- burI without IPTG induction (lane 1); overnight IPTG induction at 15 °C (lane 2); overnight IPTG induction at 37 °C (lanes 3 and 4); soluble fraction of E. coli harboring pET28a- burI with overnight IPTG induction at 37 °C (lane 5); insoluble fraction of E. coli harboring pET28a- burI with overnight IPTG induction at 37 °C (lane 6); protein marker in kDa (lane M). The BurI protein was found to be overexpressed at approximately 25 kDa in inclusion bodies.
Figure Legend Snippet: SDS-PAGE profile of overproduction of BurI in E. coli BL21 (DE3) pLysS followed by CBB staining. SDS-PAGE analysis on (A) cell lysate and (B) on soluble and insoluble fraction of cell lysate after centrifugation at 14,000 rpm. Cell lysate of E. coli harboring pET28a- burI without IPTG induction (lane 1); overnight IPTG induction at 15 °C (lane 2); overnight IPTG induction at 37 °C (lanes 3 and 4); soluble fraction of E. coli harboring pET28a- burI with overnight IPTG induction at 37 °C (lane 5); insoluble fraction of E. coli harboring pET28a- burI with overnight IPTG induction at 37 °C (lane 6); protein marker in kDa (lane M). The BurI protein was found to be overexpressed at approximately 25 kDa in inclusion bodies.

Techniques Used: SDS Page, Staining, Centrifugation, Marker

MS analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a- burI . By comparing with the corresponding synthetic AHL standard, the mass spectra demonstrated the presence of 3-oxo-C6-HSL at m / z 214.0000. (A) Mass spectra of E. coli BL21 harboring pET28a alone (control); (B) mass spectra of non-induced E. coli BL21 harboring pET28a- burI (control); (C) mass spectra of induced E. coli BL21 harboring pET28a- burI .
Figure Legend Snippet: MS analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a- burI . By comparing with the corresponding synthetic AHL standard, the mass spectra demonstrated the presence of 3-oxo-C6-HSL at m / z 214.0000. (A) Mass spectra of E. coli BL21 harboring pET28a alone (control); (B) mass spectra of non-induced E. coli BL21 harboring pET28a- burI (control); (C) mass spectra of induced E. coli BL21 harboring pET28a- burI .

Techniques Used: Mass Spectrometry

MS analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a- burI . By comparing with the corresponding synthetic AHL standard, the mass spectra demonstrated the presence of 3-hydroxy-C8-HSL at m / z 244.0000. (A) Mass spectra of E. coli BL21 harboring pET28a alone (control); (B) mass spectra of non-induced E. coli BL21 harboring pET28a- burI (control); (C) mass spectra of induced E. coli BL21 harboring pET28a- burI .
Figure Legend Snippet: MS analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a- burI . By comparing with the corresponding synthetic AHL standard, the mass spectra demonstrated the presence of 3-hydroxy-C8-HSL at m / z 244.0000. (A) Mass spectra of E. coli BL21 harboring pET28a alone (control); (B) mass spectra of non-induced E. coli BL21 harboring pET28a- burI (control); (C) mass spectra of induced E. coli BL21 harboring pET28a- burI .

Techniques Used: Mass Spectrometry

MS analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a- burI . By comparing with the corresponding synthetic AHL standard, the mass spectra demonstrated the presence of C8-HSL at m / z 228.3000. (A) Mass spectra of E. coli BL21 harboring pET28a alone (control); (B) mass spectra of non-induced E. coli BL21 harboring pET28a- burI (control); (C) mass spectra of induced E. coli BL21 harboring pET28a- burI .
Figure Legend Snippet: MS analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a- burI . By comparing with the corresponding synthetic AHL standard, the mass spectra demonstrated the presence of C8-HSL at m / z 228.3000. (A) Mass spectra of E. coli BL21 harboring pET28a alone (control); (B) mass spectra of non-induced E. coli BL21 harboring pET28a- burI (control); (C) mass spectra of induced E. coli BL21 harboring pET28a- burI .

Techniques Used: Mass Spectrometry

2) Product Images from "Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein"

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

Journal: Iranian Journal of Biotechnology

doi: 10.21859/ijb.1482

Coomassie stained SDS-PAGE analysis of cell lysates from α-Luffin producing E. coli BL21 (DE3) clones in different temperatures, time and mode of culture. Panel A: LB batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates before IPTG induction; Lane 3, Cell lysates 4h post induction; Lane 4, Cell lysates 6h post induction; Lane 5, Cell lysates 8h post induction; Lane 6, Cell lysates 12h post induction; Lane 7, Cell lysates 24h post induction; Lane 8, Protein marker SM0671. Panel B: EB Fed-batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Protein marker SM0671; Lane 3, Cell lysates before IPTG induction; Lane 4, Cell lysates 4h post induction; Lane 5, Cell lysates 6h post induction; Lane 6, Cell lysates 8h post induction; Lane 7, Cell lysates total protein 10h post induction; Lane 8, Cell lysates total protein 12h post induction; Lane 9, Cell lysates total protein 24h post induction. Panel C: LB batch culture mode in 25°C. Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates total protein before IPTG induction; Lane 3, Cell lysates total protein 4h post induction; Lane 4, Cell lysates total protein 6h post induction; Lane 5, Cell lysates total protein 8h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker SM0671. Panel D: EB Fed-batch culture mode in 25 °C . Lane1, Cell lysates total protein before IPTG induction; Lane 2, Cell lysates total protein 4h post induction; Lane 3, Cell lysates total protein 6h post induction; Lane 4, Cell lysates total protein 8h post induction; Lane 5, Cell lysates total protein 10h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker Thermo science #26610. All samples were diluted to equal cell concentration before lysis and loading on gel. The position of 28.8kDa His-α-Luffin is indicated by arrows.
Figure Legend Snippet: Coomassie stained SDS-PAGE analysis of cell lysates from α-Luffin producing E. coli BL21 (DE3) clones in different temperatures, time and mode of culture. Panel A: LB batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates before IPTG induction; Lane 3, Cell lysates 4h post induction; Lane 4, Cell lysates 6h post induction; Lane 5, Cell lysates 8h post induction; Lane 6, Cell lysates 12h post induction; Lane 7, Cell lysates 24h post induction; Lane 8, Protein marker SM0671. Panel B: EB Fed-batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Protein marker SM0671; Lane 3, Cell lysates before IPTG induction; Lane 4, Cell lysates 4h post induction; Lane 5, Cell lysates 6h post induction; Lane 6, Cell lysates 8h post induction; Lane 7, Cell lysates total protein 10h post induction; Lane 8, Cell lysates total protein 12h post induction; Lane 9, Cell lysates total protein 24h post induction. Panel C: LB batch culture mode in 25°C. Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates total protein before IPTG induction; Lane 3, Cell lysates total protein 4h post induction; Lane 4, Cell lysates total protein 6h post induction; Lane 5, Cell lysates total protein 8h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker SM0671. Panel D: EB Fed-batch culture mode in 25 °C . Lane1, Cell lysates total protein before IPTG induction; Lane 2, Cell lysates total protein 4h post induction; Lane 3, Cell lysates total protein 6h post induction; Lane 4, Cell lysates total protein 8h post induction; Lane 5, Cell lysates total protein 10h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker Thermo science #26610. All samples were diluted to equal cell concentration before lysis and loading on gel. The position of 28.8kDa His-α-Luffin is indicated by arrows.

Techniques Used: Staining, SDS Page, Marker, Concentration Assay, Lysis

Western blot analysis of total cell lysates and purified α-Luffin. A: Western blot analysis on total cell lysates of α-Luffin producing E. coli BL21 clones cultured in the fed-batch mode. Proteins were visualized with an anti-His antibody conjugated with alkaline phosphatase and DAB substrate. Lane 1, Control positive, an 18kDa His-tagged protein; Lane 2, protein marker Thermo science #26616; Lanes 3 and 4, E. coli BL21 cell lysates 6h after induction; Lane 5, Control negative before induction; Lane 6, Control negative E. coli /pET28a alone. B: SDS-PAGE (Lanes 1-8) and Western blot (Lanes 9-12) analysis of purified α-Luffin from fed batch process. Lane 1, Fed-batch soluble fraction (initial sample); Lane 2, NTA flow through sample; Lanes 3-7, Elution fractions from NTA column representing purified α-Luffin; Lane 9, His-tagged control protein (18 kDa); Lanes 11 and 12, NTA purified α-Luffin from fed-batch soluble fraction; Lanes 8 and 10, Protein size marker Thermo science #26610.
Figure Legend Snippet: Western blot analysis of total cell lysates and purified α-Luffin. A: Western blot analysis on total cell lysates of α-Luffin producing E. coli BL21 clones cultured in the fed-batch mode. Proteins were visualized with an anti-His antibody conjugated with alkaline phosphatase and DAB substrate. Lane 1, Control positive, an 18kDa His-tagged protein; Lane 2, protein marker Thermo science #26616; Lanes 3 and 4, E. coli BL21 cell lysates 6h after induction; Lane 5, Control negative before induction; Lane 6, Control negative E. coli /pET28a alone. B: SDS-PAGE (Lanes 1-8) and Western blot (Lanes 9-12) analysis of purified α-Luffin from fed batch process. Lane 1, Fed-batch soluble fraction (initial sample); Lane 2, NTA flow through sample; Lanes 3-7, Elution fractions from NTA column representing purified α-Luffin; Lane 9, His-tagged control protein (18 kDa); Lanes 11 and 12, NTA purified α-Luffin from fed-batch soluble fraction; Lanes 8 and 10, Protein size marker Thermo science #26610.

Techniques Used: Western Blot, Purification, Clone Assay, Cell Culture, Marker, SDS Page, Flow Cytometry

Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).
Figure Legend Snippet: Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).

Techniques Used: Construct, Nucleic Acid Electrophoresis, Plasmid Preparation, Marker

3) Product Images from "Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein"

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

Journal: Iranian Journal of Biotechnology

doi: 10.21859/ijb.1482

Coomassie stained SDS-PAGE analysis of cell lysates from α-Luffin producing E. coli BL21 (DE3) clones in different temperatures, time and mode of culture. Panel A: LB batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates before IPTG induction; Lane 3, Cell lysates 4h post induction; Lane 4, Cell lysates 6h post induction; Lane 5, Cell lysates 8h post induction; Lane 6, Cell lysates 12h post induction; Lane 7, Cell lysates 24h post induction; Lane 8, Protein marker SM0671. Panel B: EB Fed-batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Protein marker SM0671; Lane 3, Cell lysates before IPTG induction; Lane 4, Cell lysates 4h post induction; Lane 5, Cell lysates 6h post induction; Lane 6, Cell lysates 8h post induction; Lane 7, Cell lysates total protein 10h post induction; Lane 8, Cell lysates total protein 12h post induction; Lane 9, Cell lysates total protein 24h post induction. Panel C: LB batch culture mode in 25°C. Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates total protein before IPTG induction; Lane 3, Cell lysates total protein 4h post induction; Lane 4, Cell lysates total protein 6h post induction; Lane 5, Cell lysates total protein 8h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker SM0671. Panel D: EB Fed-batch culture mode in 25 °C . Lane1, Cell lysates total protein before IPTG induction; Lane 2, Cell lysates total protein 4h post induction; Lane 3, Cell lysates total protein 6h post induction; Lane 4, Cell lysates total protein 8h post induction; Lane 5, Cell lysates total protein 10h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker Thermo science #26610. All samples were diluted to equal cell concentration before lysis and loading on gel. The position of 28.8kDa His-α-Luffin is indicated by arrows.
Figure Legend Snippet: Coomassie stained SDS-PAGE analysis of cell lysates from α-Luffin producing E. coli BL21 (DE3) clones in different temperatures, time and mode of culture. Panel A: LB batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates before IPTG induction; Lane 3, Cell lysates 4h post induction; Lane 4, Cell lysates 6h post induction; Lane 5, Cell lysates 8h post induction; Lane 6, Cell lysates 12h post induction; Lane 7, Cell lysates 24h post induction; Lane 8, Protein marker SM0671. Panel B: EB Fed-batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Protein marker SM0671; Lane 3, Cell lysates before IPTG induction; Lane 4, Cell lysates 4h post induction; Lane 5, Cell lysates 6h post induction; Lane 6, Cell lysates 8h post induction; Lane 7, Cell lysates total protein 10h post induction; Lane 8, Cell lysates total protein 12h post induction; Lane 9, Cell lysates total protein 24h post induction. Panel C: LB batch culture mode in 25°C. Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates total protein before IPTG induction; Lane 3, Cell lysates total protein 4h post induction; Lane 4, Cell lysates total protein 6h post induction; Lane 5, Cell lysates total protein 8h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker SM0671. Panel D: EB Fed-batch culture mode in 25 °C . Lane1, Cell lysates total protein before IPTG induction; Lane 2, Cell lysates total protein 4h post induction; Lane 3, Cell lysates total protein 6h post induction; Lane 4, Cell lysates total protein 8h post induction; Lane 5, Cell lysates total protein 10h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker Thermo science #26610. All samples were diluted to equal cell concentration before lysis and loading on gel. The position of 28.8kDa His-α-Luffin is indicated by arrows.

Techniques Used: Staining, SDS Page, Marker, Concentration Assay, Lysis

Western blot analysis of total cell lysates and purified α-Luffin. A: Western blot analysis on total cell lysates of α-Luffin producing E. coli BL21 clones cultured in the fed-batch mode. Proteins were visualized with an anti-His antibody conjugated with alkaline phosphatase and DAB substrate. Lane 1, Control positive, an 18kDa His-tagged protein; Lane 2, protein marker Thermo science #26616; Lanes 3 and 4, E. coli BL21 cell lysates 6h after induction; Lane 5, Control negative before induction; Lane 6, Control negative E. coli /pET28a alone. B: SDS-PAGE (Lanes 1-8) and Western blot (Lanes 9-12) analysis of purified α-Luffin from fed batch process. Lane 1, Fed-batch soluble fraction (initial sample); Lane 2, NTA flow through sample; Lanes 3-7, Elution fractions from NTA column representing purified α-Luffin; Lane 9, His-tagged control protein (18 kDa); Lanes 11 and 12, NTA purified α-Luffin from fed-batch soluble fraction; Lanes 8 and 10, Protein size marker Thermo science #26610.
Figure Legend Snippet: Western blot analysis of total cell lysates and purified α-Luffin. A: Western blot analysis on total cell lysates of α-Luffin producing E. coli BL21 clones cultured in the fed-batch mode. Proteins were visualized with an anti-His antibody conjugated with alkaline phosphatase and DAB substrate. Lane 1, Control positive, an 18kDa His-tagged protein; Lane 2, protein marker Thermo science #26616; Lanes 3 and 4, E. coli BL21 cell lysates 6h after induction; Lane 5, Control negative before induction; Lane 6, Control negative E. coli /pET28a alone. B: SDS-PAGE (Lanes 1-8) and Western blot (Lanes 9-12) analysis of purified α-Luffin from fed batch process. Lane 1, Fed-batch soluble fraction (initial sample); Lane 2, NTA flow through sample; Lanes 3-7, Elution fractions from NTA column representing purified α-Luffin; Lane 9, His-tagged control protein (18 kDa); Lanes 11 and 12, NTA purified α-Luffin from fed-batch soluble fraction; Lanes 8 and 10, Protein size marker Thermo science #26610.

Techniques Used: Western Blot, Purification, Clone Assay, Cell Culture, Marker, SDS Page, Flow Cytometry

Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).
Figure Legend Snippet: Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).

Techniques Used: Construct, Nucleic Acid Electrophoresis, Plasmid Preparation, Marker

4) Product Images from "An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli"

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1197

Effect of TopAI on DNA topology. ( A ) E. coli BL21(DE3) harboring pET28a or pET- topAI was grown at 37°C in M9-glucose liquid medium. When O.D. 600 reached at 0.6, IPTG was added to a final concentration of 0.1 mM. At the different time intervals indicated, 50 ml of the culture were removed and DNA topology of plasmid was analyzed. These analyses were carried out in the presence of 2.5 μg/ml chloroquine. ( B ) E. coli BL21(DE3) harboring pET- topAI was grown at 37°C in M9-glucose liquid medium. When O.D. 600 reached at 0.6, different concentration of IPTG was added (lane 2; 0 mM, lane 3; 0.01 mM, lane 4; 0.1 mM and lane 5; 1 mM). After 1 h incubation, 50 ml of the culture were removed and DNA topology of the plasmids was analyzed as described in Materials and Methods. E. coli BL21(DE3) containing pET28a was incubated with 1 mM IPTG and the extracted plasmid was analyzed as negative control (lane 1).
Figure Legend Snippet: Effect of TopAI on DNA topology. ( A ) E. coli BL21(DE3) harboring pET28a or pET- topAI was grown at 37°C in M9-glucose liquid medium. When O.D. 600 reached at 0.6, IPTG was added to a final concentration of 0.1 mM. At the different time intervals indicated, 50 ml of the culture were removed and DNA topology of plasmid was analyzed. These analyses were carried out in the presence of 2.5 μg/ml chloroquine. ( B ) E. coli BL21(DE3) harboring pET- topAI was grown at 37°C in M9-glucose liquid medium. When O.D. 600 reached at 0.6, different concentration of IPTG was added (lane 2; 0 mM, lane 3; 0.01 mM, lane 4; 0.1 mM and lane 5; 1 mM). After 1 h incubation, 50 ml of the culture were removed and DNA topology of the plasmids was analyzed as described in Materials and Methods. E. coli BL21(DE3) containing pET28a was incubated with 1 mM IPTG and the extracted plasmid was analyzed as negative control (lane 1).

Techniques Used: Positron Emission Tomography, Concentration Assay, Plasmid Preparation, Incubation, Negative Control

Identification of TopAI and YjhQ as a TA system. ( A ) E. coli BL21 transformed with pET28a and pBAD24 or pET- topAI and pBAD- yjhQ was streaked on M9 (glycerol, CAA) plates with 0.1 mM IPTG, 0.2% arabinose, 0.1 mM IPTG plus 0.2% arabinose or without both inducers. The plates were incubated at 37°C for 18 h. ( B ) Interaction between TopAI and YjhQ in a pull-down assay. Purified PrS-TopAI or PrS containing a His-tag and PrS 2 -YjhQ containing Strep-tag were incubated at 4°C for 1 h. The complex was recovered by nickel-resin. The mixture (lanes 1 and 6), flow-through (lanes 2 and 7), wash fraction (lanes 3 and 8) and elution fraction (lanes 4, 5 and 9, 10) were analyzed by western blotting using His-tag antibody or Strep-tag antibody. ( C ) Growth curves of E. coli BL21(DE3) harboring pET- topAI . The cells were cultured in M9-glucose liquid medium at 37°C in the presence (closed circles) or absence (open circles) of 0.1 mM IPTG. ( D ) Colony formation units after induction of TopAI. E. coli BL21(DE3) harboring pET- topAI was cultured in M9-glucose. When O.D. reached 0.6, 0.1 mM IPTG was added. The cells were collected, washed three times with saline and spread on M9-glucose plates. The plates were incubated at 37°C for 18 h and the number of colony was counted. ( E ) Alignment of E. coli TopAI with other TopAI homologues from Salmonella typhimurium , Pseudomonas aeruginosa , Caulobacter crescentus and Myxococcus xanthus . Identical amino acid residues are shown in black shades and conservative substitutions in gray shades.
Figure Legend Snippet: Identification of TopAI and YjhQ as a TA system. ( A ) E. coli BL21 transformed with pET28a and pBAD24 or pET- topAI and pBAD- yjhQ was streaked on M9 (glycerol, CAA) plates with 0.1 mM IPTG, 0.2% arabinose, 0.1 mM IPTG plus 0.2% arabinose or without both inducers. The plates were incubated at 37°C for 18 h. ( B ) Interaction between TopAI and YjhQ in a pull-down assay. Purified PrS-TopAI or PrS containing a His-tag and PrS 2 -YjhQ containing Strep-tag were incubated at 4°C for 1 h. The complex was recovered by nickel-resin. The mixture (lanes 1 and 6), flow-through (lanes 2 and 7), wash fraction (lanes 3 and 8) and elution fraction (lanes 4, 5 and 9, 10) were analyzed by western blotting using His-tag antibody or Strep-tag antibody. ( C ) Growth curves of E. coli BL21(DE3) harboring pET- topAI . The cells were cultured in M9-glucose liquid medium at 37°C in the presence (closed circles) or absence (open circles) of 0.1 mM IPTG. ( D ) Colony formation units after induction of TopAI. E. coli BL21(DE3) harboring pET- topAI was cultured in M9-glucose. When O.D. reached 0.6, 0.1 mM IPTG was added. The cells were collected, washed three times with saline and spread on M9-glucose plates. The plates were incubated at 37°C for 18 h and the number of colony was counted. ( E ) Alignment of E. coli TopAI with other TopAI homologues from Salmonella typhimurium , Pseudomonas aeruginosa , Caulobacter crescentus and Myxococcus xanthus . Identical amino acid residues are shown in black shades and conservative substitutions in gray shades.

Techniques Used: Transformation Assay, Positron Emission Tomography, Cellular Antioxidant Activity Assay, Incubation, Pull Down Assay, Purification, Strep-tag, Flow Cytometry, Western Blot, Cell Culture

5) Product Images from "Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region"

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region

Journal: Infection and Immunity

doi:

Time course of expression of a conserved region of human P. carinii MSG 33, as evaluated by SDS-PAGE (14% gel) and Coomassie blue staining. (A) Expression of recombinant protein (arrow) at different time points after induction with IPTG (1 mM). Maximal expression of the protein can be reached at 2 h (arrow). (B) The pET28A vector alone under the same conditions. No equivalent band is seen.
Figure Legend Snippet: Time course of expression of a conserved region of human P. carinii MSG 33, as evaluated by SDS-PAGE (14% gel) and Coomassie blue staining. (A) Expression of recombinant protein (arrow) at different time points after induction with IPTG (1 mM). Maximal expression of the protein can be reached at 2 h (arrow). (B) The pET28A vector alone under the same conditions. No equivalent band is seen.

Techniques Used: Expressing, SDS Page, Staining, Recombinant, Plasmid Preparation

Immunoblots with a recombinant conserved human P. carinii MSG peptide. (A) Lane 1, reactivity of the T7-tag monoclonal antibody, which reacts with a pET28A vector-derived epitope that precedes the MSG peptide (arrow); lane 2, reactivity with a polyclonal anti-epitope antibody generated against a conserved epitope contained within the recombinant MSG fragment; lanes 3 and 4, reactivity with serum samples from healthy humans (diluted 1:100). (B) Reactivities of a variety of human serum samples (diluted 1:100). Lanes 1, 4 and 6, serum samples from healthy humans; lanes 2 and 7, serum samples from HIV-infected patients with a history of P. carinii pneumonia; lanes 3, 5, and 8, serum samples from patients with a history of P. carinii pneumonia but without HIV infection; lane 9, serum from an immunosuppressed patient without P. carinii pneumonia or HIV infection. All samples reacted by immunoblotting with the recombinant peptide (arrow). (C) Reactivity with no first antibody (lane 1) or reactivity of serum (diluted 1:100) from a cat, mouse, or rat (lanes 2 to 4, respectively). For lane 1, the second antibody was alkaline phosphatase-conjugated goat anti-human antibody; for each of the remaining lanes, the appropriate alkaline phosphatase-conjugated second antibody was used.
Figure Legend Snippet: Immunoblots with a recombinant conserved human P. carinii MSG peptide. (A) Lane 1, reactivity of the T7-tag monoclonal antibody, which reacts with a pET28A vector-derived epitope that precedes the MSG peptide (arrow); lane 2, reactivity with a polyclonal anti-epitope antibody generated against a conserved epitope contained within the recombinant MSG fragment; lanes 3 and 4, reactivity with serum samples from healthy humans (diluted 1:100). (B) Reactivities of a variety of human serum samples (diluted 1:100). Lanes 1, 4 and 6, serum samples from healthy humans; lanes 2 and 7, serum samples from HIV-infected patients with a history of P. carinii pneumonia; lanes 3, 5, and 8, serum samples from patients with a history of P. carinii pneumonia but without HIV infection; lane 9, serum from an immunosuppressed patient without P. carinii pneumonia or HIV infection. All samples reacted by immunoblotting with the recombinant peptide (arrow). (C) Reactivity with no first antibody (lane 1) or reactivity of serum (diluted 1:100) from a cat, mouse, or rat (lanes 2 to 4, respectively). For lane 1, the second antibody was alkaline phosphatase-conjugated goat anti-human antibody; for each of the remaining lanes, the appropriate alkaline phosphatase-conjugated second antibody was used.

Techniques Used: Western Blot, Recombinant, Plasmid Preparation, Derivative Assay, Generated, Infection

6) Product Images from "High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development"

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-10-56

VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and pGEX-4T-1 (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.
Figure Legend Snippet: VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and pGEX-4T-1 (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

Techniques Used: Expressing, SDS Page, Western Blot, Staining, Marker

Schematic diagram of the constructs used for CVA VP1 protein expression . (A) Schematic representation of the VP1 protein variants and expression vectors used in this study. The designations of the VP1 protein and its expression vectors are indicated, a, b and c. The first two constructs, a and b, contained the full-length VP1 gene cloned into vectors pET28a and pGEX-4T-1, for expression of VP1 protein with a six-histidine (6 × His) tag and glutathione-s-transferase (GST) tag at its N-terminus, respectively. Construct c contained VP1 gene with codon-optimized at its N-terminus; this was derived from construct b by replacing the rare codons in the area 1-321 bp without altering amino acid sequence. The N-terminal codon-optimized VP1 gene, opt-N, was fused with C-terminal domain of VP1 gene (VP1 C-term) by overlapping PCR, and then cloned into pGEX-4T-1. (B) Sequence comparison between the WT VP1 and Opt VP1 gene. The nucleotide sequences were compared between the original VP1 gene (WT VP1) and the sequence of codon-optimized VP1 gene (Opt VP1) over the N-terminal region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: Schematic diagram of the constructs used for CVA VP1 protein expression . (A) Schematic representation of the VP1 protein variants and expression vectors used in this study. The designations of the VP1 protein and its expression vectors are indicated, a, b and c. The first two constructs, a and b, contained the full-length VP1 gene cloned into vectors pET28a and pGEX-4T-1, for expression of VP1 protein with a six-histidine (6 × His) tag and glutathione-s-transferase (GST) tag at its N-terminus, respectively. Construct c contained VP1 gene with codon-optimized at its N-terminus; this was derived from construct b by replacing the rare codons in the area 1-321 bp without altering amino acid sequence. The N-terminal codon-optimized VP1 gene, opt-N, was fused with C-terminal domain of VP1 gene (VP1 C-term) by overlapping PCR, and then cloned into pGEX-4T-1. (B) Sequence comparison between the WT VP1 and Opt VP1 gene. The nucleotide sequences were compared between the original VP1 gene (WT VP1) and the sequence of codon-optimized VP1 gene (Opt VP1) over the N-terminal region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.

Techniques Used: Construct, Expressing, Clone Assay, Derivative Assay, Sequencing, Polymerase Chain Reaction

7) Product Images from "Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene"

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00240

Analysis of aciI gene and protein. (A) Ethidium bromide-stained agarose gel containing aciI (gene amplification by PCR). Lanes 1 and 2 shows the amplified 552 bp amplicon. 5 μl of PCR products were loaded into each lane and electrophoresis was performed at 100 V. (B) SDS-PAGE analysis of the purified recombinant AciI protein. Lane 3, cell lysates of non-induced E. coli BL21 harboring pET28a-aciI; Lane 4, cell lysates of induced E. coli BL21 harboring pET28a-aciI; lane 5, flow-through fraction of purification step; lane 6, wash fraction of purification step; lane 7, eluted fraction containing recombinant AciI protein; lane M1, 1 kb DNA marker (Fermentas, Thermo Fisher Scientific, USA); lane M2, molecular weight markers (Bio-Rad, USA) with mass of each marker protein in kDa as indicated. The same amount of protein was loaded into each lane and subjected to electrophoresis at 150 V.
Figure Legend Snippet: Analysis of aciI gene and protein. (A) Ethidium bromide-stained agarose gel containing aciI (gene amplification by PCR). Lanes 1 and 2 shows the amplified 552 bp amplicon. 5 μl of PCR products were loaded into each lane and electrophoresis was performed at 100 V. (B) SDS-PAGE analysis of the purified recombinant AciI protein. Lane 3, cell lysates of non-induced E. coli BL21 harboring pET28a-aciI; Lane 4, cell lysates of induced E. coli BL21 harboring pET28a-aciI; lane 5, flow-through fraction of purification step; lane 6, wash fraction of purification step; lane 7, eluted fraction containing recombinant AciI protein; lane M1, 1 kb DNA marker (Fermentas, Thermo Fisher Scientific, USA); lane M2, molecular weight markers (Bio-Rad, USA) with mass of each marker protein in kDa as indicated. The same amount of protein was loaded into each lane and subjected to electrophoresis at 150 V.

Techniques Used: Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Electrophoresis, SDS Page, Purification, Recombinant, Flow Cytometry, Marker, Molecular Weight

Mass spectrometry (MS) analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a-aciI showing the presence of 3-oxo-C12-HSL and 3-hydroxy-C12-HSL. By comparing with the corresponding synthetic AHL standards, the mass spectra demonstrated the presence of (A) 3-oxo-C12-HSL at m/z 298.1000 and (B) 3-hydroxy-C12-HSL at m/z 300.1000. The retention time for 3-oxo-C12-HSL and 3-hydroxy-C12-HSL are 6.684 min and 5.867 min, respectively. (i) Mass spectra of E. coli BL21 harboring pET28a alone (control); (ii) mass spectra of non-induced E. coli BL21 harboring pET28a-aciI (control); (iii) mass spectra of induced E. coli BL21 harboring pET28a-aciI.
Figure Legend Snippet: Mass spectrometry (MS) analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a-aciI showing the presence of 3-oxo-C12-HSL and 3-hydroxy-C12-HSL. By comparing with the corresponding synthetic AHL standards, the mass spectra demonstrated the presence of (A) 3-oxo-C12-HSL at m/z 298.1000 and (B) 3-hydroxy-C12-HSL at m/z 300.1000. The retention time for 3-oxo-C12-HSL and 3-hydroxy-C12-HSL are 6.684 min and 5.867 min, respectively. (i) Mass spectra of E. coli BL21 harboring pET28a alone (control); (ii) mass spectra of non-induced E. coli BL21 harboring pET28a-aciI (control); (iii) mass spectra of induced E. coli BL21 harboring pET28a-aciI.

Techniques Used: Mass Spectrometry

8) Product Images from "Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network"

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network

Journal: Scientific Reports

doi: 10.1038/srep24255

HPLC analyses of TcsA-ACP ( A ), stw ACP ( B ), and sts FAS ACP ( C ). Each of ACP genes was cloned into pET28a. Each of PPTase genes was cloned into pYY0040, a plasmid derived from pET44a. E. coli strains harboring both a ACP -containing-plasmid and a PPTase -containing-plasmid were induced with IPTG to produce His-tagged ACPs with intact PPTases. His-tagged ACPs were purified by affinity chromatograph and then analyzed by HPLC and MS. ▽, apo-form; ▼, holo-form.
Figure Legend Snippet: HPLC analyses of TcsA-ACP ( A ), stw ACP ( B ), and sts FAS ACP ( C ). Each of ACP genes was cloned into pET28a. Each of PPTase genes was cloned into pYY0040, a plasmid derived from pET44a. E. coli strains harboring both a ACP -containing-plasmid and a PPTase -containing-plasmid were induced with IPTG to produce His-tagged ACPs with intact PPTases. His-tagged ACPs were purified by affinity chromatograph and then analyzed by HPLC and MS. ▽, apo-form; ▼, holo-form.

Techniques Used: High Performance Liquid Chromatography, Clone Assay, Plasmid Preparation, Derivative Assay, Purification, Mass Spectrometry

MS analyses of FkbP-PCP. FkbP-PCP gene fused with SUMO gene was cloned into pET28a. Each of PPTase genes was cloned into pYY0040. E. coli strains harboring both a PCP -containing-plasmid and a PPTase -containing-plasmid were induced with IPTG to produce His-tagged PCPs with intact PPTases. His-tagged PCPs were purified by affinity chromatograph and then analyzed by HPLC and MS. The calculated molecular weights of apo-form and holo-form of FkbP-PCP is 26,970 Da and 27,310 Da, respectively.
Figure Legend Snippet: MS analyses of FkbP-PCP. FkbP-PCP gene fused with SUMO gene was cloned into pET28a. Each of PPTase genes was cloned into pYY0040. E. coli strains harboring both a PCP -containing-plasmid and a PPTase -containing-plasmid were induced with IPTG to produce His-tagged PCPs with intact PPTases. His-tagged PCPs were purified by affinity chromatograph and then analyzed by HPLC and MS. The calculated molecular weights of apo-form and holo-form of FkbP-PCP is 26,970 Da and 27,310 Da, respectively.

Techniques Used: Mass Spectrometry, Clone Assay, Plasmid Preparation, Purification, High Performance Liquid Chromatography

9) Product Images from "Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri"

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00072

Mass spectra showing the AHL profile of spent culture supernatant of E. coli BL21(DE3)pLysS harboring pET28a_ cneI . ACN (A) was used as blank. The EIC spectra of E. coli with pET_28a alone (B) and with pET28a_ cneI (C) were compared with that of synthetic AHL, C4-HSL (D) at the same retention time. The detection of the peaks with m/z 172.100 signify the presence of C4-HSL as shown by arrows.
Figure Legend Snippet: Mass spectra showing the AHL profile of spent culture supernatant of E. coli BL21(DE3)pLysS harboring pET28a_ cneI . ACN (A) was used as blank. The EIC spectra of E. coli with pET_28a alone (B) and with pET28a_ cneI (C) were compared with that of synthetic AHL, C4-HSL (D) at the same retention time. The detection of the peaks with m/z 172.100 signify the presence of C4-HSL as shown by arrows.

Techniques Used: Positron Emission Tomography

The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of CneI overexpression (Lanes 1–5) and purification (Lanes 6–8) from E. coli BL21(DE3)pLysS. Lane 1: Cell lysate of E. coli BL21(DE3)pLysS. Lanes 2 and 3: Cell lysate of E. coli BL21(DE3)pLysS transformed with empty pET28a with and without IPTG induction, respectively. Lanes 4 and 5: Cell lysates of E. coli BL21(DE3)pLysS harboring pET28a_ cneI with and without induction, respectively. Lane 6: Flowthrough fraction. Lane 7: Wash fraction. Lane 8: Eluted fraction. Lane 9: PageRuler prestained protein ladder in kiloDalton (kDa). (Thermo Scientific, Waltham, MA, USA).
Figure Legend Snippet: The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of CneI overexpression (Lanes 1–5) and purification (Lanes 6–8) from E. coli BL21(DE3)pLysS. Lane 1: Cell lysate of E. coli BL21(DE3)pLysS. Lanes 2 and 3: Cell lysate of E. coli BL21(DE3)pLysS transformed with empty pET28a with and without IPTG induction, respectively. Lanes 4 and 5: Cell lysates of E. coli BL21(DE3)pLysS harboring pET28a_ cneI with and without induction, respectively. Lane 6: Flowthrough fraction. Lane 7: Wash fraction. Lane 8: Eluted fraction. Lane 9: PageRuler prestained protein ladder in kiloDalton (kDa). (Thermo Scientific, Waltham, MA, USA).

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Over Expression, Purification, Transformation Assay

10) Product Images from "Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region"

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region

Journal: Infection and Immunity

doi:

Time course of expression of a conserved region of human P. carinii MSG 33, as evaluated by SDS-PAGE (14% gel) and Coomassie blue staining. (A) Expression of recombinant protein (arrow) at different time points after induction with IPTG (1 mM). Maximal expression of the protein can be reached at 2 h (arrow). (B) The pET28A vector alone under the same conditions. No equivalent band is seen.
Figure Legend Snippet: Time course of expression of a conserved region of human P. carinii MSG 33, as evaluated by SDS-PAGE (14% gel) and Coomassie blue staining. (A) Expression of recombinant protein (arrow) at different time points after induction with IPTG (1 mM). Maximal expression of the protein can be reached at 2 h (arrow). (B) The pET28A vector alone under the same conditions. No equivalent band is seen.

Techniques Used: Expressing, SDS Page, Staining, Recombinant, Plasmid Preparation

Immunoblots with a recombinant conserved human P. carinii MSG peptide. (A) Lane 1, reactivity of the T7-tag monoclonal antibody, which reacts with a pET28A vector-derived epitope that precedes the MSG peptide (arrow); lane 2, reactivity with a polyclonal anti-epitope antibody generated against a conserved epitope contained within the recombinant MSG fragment; lanes 3 and 4, reactivity with serum samples from healthy humans (diluted 1:100). (B) Reactivities of a variety of human serum samples (diluted 1:100). Lanes 1, 4 and 6, serum samples from healthy humans; lanes 2 and 7, serum samples from HIV-infected patients with a history of P. carinii pneumonia; lanes 3, 5, and 8, serum samples from patients with a history of P. carinii pneumonia but without HIV infection; lane 9, serum from an immunosuppressed patient without P. carinii pneumonia or HIV infection. All samples reacted by immunoblotting with the recombinant peptide (arrow). (C) Reactivity with no first antibody (lane 1) or reactivity of serum (diluted 1:100) from a cat, mouse, or rat (lanes 2 to 4, respectively). For lane 1, the second antibody was alkaline phosphatase-conjugated goat anti-human antibody; for each of the remaining lanes, the appropriate alkaline phosphatase-conjugated second antibody was used.
Figure Legend Snippet: Immunoblots with a recombinant conserved human P. carinii MSG peptide. (A) Lane 1, reactivity of the T7-tag monoclonal antibody, which reacts with a pET28A vector-derived epitope that precedes the MSG peptide (arrow); lane 2, reactivity with a polyclonal anti-epitope antibody generated against a conserved epitope contained within the recombinant MSG fragment; lanes 3 and 4, reactivity with serum samples from healthy humans (diluted 1:100). (B) Reactivities of a variety of human serum samples (diluted 1:100). Lanes 1, 4 and 6, serum samples from healthy humans; lanes 2 and 7, serum samples from HIV-infected patients with a history of P. carinii pneumonia; lanes 3, 5, and 8, serum samples from patients with a history of P. carinii pneumonia but without HIV infection; lane 9, serum from an immunosuppressed patient without P. carinii pneumonia or HIV infection. All samples reacted by immunoblotting with the recombinant peptide (arrow). (C) Reactivity with no first antibody (lane 1) or reactivity of serum (diluted 1:100) from a cat, mouse, or rat (lanes 2 to 4, respectively). For lane 1, the second antibody was alkaline phosphatase-conjugated goat anti-human antibody; for each of the remaining lanes, the appropriate alkaline phosphatase-conjugated second antibody was used.

Techniques Used: Western Blot, Recombinant, Plasmid Preparation, Derivative Assay, Generated, Infection

11) Product Images from "The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas"

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Journal: Cellular Signalling

doi: 10.1016/j.cellsig.2013.10.008

Heterologous expression and characterization of Acas RegA . A/B. Heterologous expression. The Acas RegA cDNA was fused to a hexa-his tag in vector pET28a, expressed in E.coli, and purified by Ni + chromatography. The column flow-through (FT) and three fractions eluted with 250 mM imidazole were size-fractionated by SDS-PAGE (A). Western blots of the size-fractionated proteins (B) were incubated with 1:2000 diluted mouse anti his-tag antibody and 1:5000 diluted peroxidase conjugated goat-anti-mouse IgG, followed by peroxidase detection. C. Acas RegA activity . 1 μl aliquots of the combined 250 mM imidazole eluate fractions of expressed Acas RegA and combined eluates obtained from the same amount of E.coli cells, transformed with empty pET28a vector, were incubated for 30 min with 10 nM 3 H-cAMP and assayed for 3 H-cAMP hydrolysis. D. Mg 2 + dependence. Purified Acas RegA was incubated with 10 nM 3 H-cAMP and increasing concentrations of MgCl 2 and assayed for 3 H-cAMP hydrolysis. Data are expressed as percentage of 3 H-cAMP hydrolysis occurring at 0.3 mM MgCl 2. E. Substrate s pecificity. Purified Acas RegA was incubated with 10 nM 3 H-cAMP and increasing concentrations of cAMP and cGMP, and assayed for 3 H-cAMP hydrolysis. Data are expressed as percentage of hydrolysis at 10 nM 3 H-cAMP only. Means and SD of two experiments performed in triplicate are presented. F. The data for competition by cAMP (panel E) were converted into moles of 5′AMP produced per μg protein per min (V) at each concentration (S) and plotted as S/V against S in a Hanes plot. Intersections of the plot with the abscissa and ordinate, represent − K M and K M /V max values, respectively, yielding a K M of 19 μM and a V max of 55 nmol/min μg protein.
Figure Legend Snippet: Heterologous expression and characterization of Acas RegA . A/B. Heterologous expression. The Acas RegA cDNA was fused to a hexa-his tag in vector pET28a, expressed in E.coli, and purified by Ni + chromatography. The column flow-through (FT) and three fractions eluted with 250 mM imidazole were size-fractionated by SDS-PAGE (A). Western blots of the size-fractionated proteins (B) were incubated with 1:2000 diluted mouse anti his-tag antibody and 1:5000 diluted peroxidase conjugated goat-anti-mouse IgG, followed by peroxidase detection. C. Acas RegA activity . 1 μl aliquots of the combined 250 mM imidazole eluate fractions of expressed Acas RegA and combined eluates obtained from the same amount of E.coli cells, transformed with empty pET28a vector, were incubated for 30 min with 10 nM 3 H-cAMP and assayed for 3 H-cAMP hydrolysis. D. Mg 2 + dependence. Purified Acas RegA was incubated with 10 nM 3 H-cAMP and increasing concentrations of MgCl 2 and assayed for 3 H-cAMP hydrolysis. Data are expressed as percentage of 3 H-cAMP hydrolysis occurring at 0.3 mM MgCl 2. E. Substrate s pecificity. Purified Acas RegA was incubated with 10 nM 3 H-cAMP and increasing concentrations of cAMP and cGMP, and assayed for 3 H-cAMP hydrolysis. Data are expressed as percentage of hydrolysis at 10 nM 3 H-cAMP only. Means and SD of two experiments performed in triplicate are presented. F. The data for competition by cAMP (panel E) were converted into moles of 5′AMP produced per μg protein per min (V) at each concentration (S) and plotted as S/V against S in a Hanes plot. Intersections of the plot with the abscissa and ordinate, represent − K M and K M /V max values, respectively, yielding a K M of 19 μM and a V max of 55 nmol/min μg protein.

Techniques Used: Expressing, Plasmid Preparation, Purification, Chromatography, Flow Cytometry, SDS Page, Western Blot, Incubation, Activity Assay, Transformation Assay, Produced, Concentration Assay

12) Product Images from "Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula"

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23040861

Recombinant protein expression and enzymatic analysis of VbDFR. ( a ) a SDS-PAGE image shows recombinant VbDFR protein induced in E. coli BL21 (DE3) plysS strain. Lane 1: 20 μg crude protein extracts from BL21 (DE3) plysS/pET28a (+) vector control; lane 2: 20 μg crude protein extracts from BL21 (DE3) plysS/pET28a (+)-VbDFR. Lane 3: insoluble crude protein extracts from BL21 (DE3) plysS/pET28a (+)-VbDFR. Lane 4: soluble crude protein extracts from BL21 (DE3) plysS/pET28a (+)-VbDFR. M: protein molecular weight marker; ( b ) an image shows recombinant VbDFR purified; ( c ) HPLC profiles show one product formed from the incubation of taxifolin and recombinant VbDFR (c-1) but not denatured VbDFR (c-2); c-3, taxifolin standard; ( d ) HPLC profiles show one product formed from the incubation of dihydrokaempferol (DHK) and recombinant VbDFR (d-1) but not denatured recombinant VbDFR (d-2); d-3 DHK standard.
Figure Legend Snippet: Recombinant protein expression and enzymatic analysis of VbDFR. ( a ) a SDS-PAGE image shows recombinant VbDFR protein induced in E. coli BL21 (DE3) plysS strain. Lane 1: 20 μg crude protein extracts from BL21 (DE3) plysS/pET28a (+) vector control; lane 2: 20 μg crude protein extracts from BL21 (DE3) plysS/pET28a (+)-VbDFR. Lane 3: insoluble crude protein extracts from BL21 (DE3) plysS/pET28a (+)-VbDFR. Lane 4: soluble crude protein extracts from BL21 (DE3) plysS/pET28a (+)-VbDFR. M: protein molecular weight marker; ( b ) an image shows recombinant VbDFR purified; ( c ) HPLC profiles show one product formed from the incubation of taxifolin and recombinant VbDFR (c-1) but not denatured VbDFR (c-2); c-3, taxifolin standard; ( d ) HPLC profiles show one product formed from the incubation of dihydrokaempferol (DHK) and recombinant VbDFR (d-1) but not denatured recombinant VbDFR (d-2); d-3 DHK standard.

Techniques Used: Recombinant, Expressing, SDS Page, Plasmid Preparation, Molecular Weight, Marker, Purification, High Performance Liquid Chromatography, Incubation

13) Product Images from "Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1"

Article Title: Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1

Journal: Journal of Bacteriology

doi: 10.1128/JB.00250-16

(A) SDS-PAGE analysis of overexpressed recombinant HndA protein. Lane 1, crude extract of E. coli BL21(DE3) carrying empty pET28a vector; lane 2, crude extracts of induced E. coli BL21(DE3) carrying pET28a:HndA; lane 3, purified recombinant HndA protein; lane M, molecular mass marker (Puregene). (B) Spectral changes during transformation of 2HINA by purified recombinant HndA protein. The sample and reference cuvettes contained 50 mM potassium phosphate buffer (pH 7.0) in a 1-ml volume. The sample cuvette also contained 220 nmol of 2H1NA. Spectra were recorded every 1 min after the addition of 10 μg of protein to both cuvettes. The up and down arrows indicate increasing and decreasing absorbances, respectively. (C) HPLC chromatogram showing transformation of 2H1NA to 2-naphthol by purified HndA in a reaction mixture (final volume, 1 ml) containing 0.5 mM 2H1NA and 5 μg of protein in buffer A incubated for 10 min at 35°C. UV-visible light spectra of 2H1NA and 2-naphthol are shown in insets. (D) Time-dependent transformation of 2H1NA to 2-naphthol by purified HndA. The concentrations of 2H1NA (○) and 2-naphthol (●) were determined by HPLC from the reaction mixtures (as described in panel C) during enzymatic transformation over 10 min.
Figure Legend Snippet: (A) SDS-PAGE analysis of overexpressed recombinant HndA protein. Lane 1, crude extract of E. coli BL21(DE3) carrying empty pET28a vector; lane 2, crude extracts of induced E. coli BL21(DE3) carrying pET28a:HndA; lane 3, purified recombinant HndA protein; lane M, molecular mass marker (Puregene). (B) Spectral changes during transformation of 2HINA by purified recombinant HndA protein. The sample and reference cuvettes contained 50 mM potassium phosphate buffer (pH 7.0) in a 1-ml volume. The sample cuvette also contained 220 nmol of 2H1NA. Spectra were recorded every 1 min after the addition of 10 μg of protein to both cuvettes. The up and down arrows indicate increasing and decreasing absorbances, respectively. (C) HPLC chromatogram showing transformation of 2H1NA to 2-naphthol by purified HndA in a reaction mixture (final volume, 1 ml) containing 0.5 mM 2H1NA and 5 μg of protein in buffer A incubated for 10 min at 35°C. UV-visible light spectra of 2H1NA and 2-naphthol are shown in insets. (D) Time-dependent transformation of 2H1NA to 2-naphthol by purified HndA. The concentrations of 2H1NA (○) and 2-naphthol (●) were determined by HPLC from the reaction mixtures (as described in panel C) during enzymatic transformation over 10 min.

Techniques Used: SDS Page, Recombinant, Plasmid Preparation, Purification, Marker, Transformation Assay, High Performance Liquid Chromatography, Incubation

14) Product Images from "Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil"

Article Title: Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02375

12% SDS–PAGE gel representing the purification of recombinant Vcz and URA3 deaminases. 1, 5, and 9: PageRuler TM Prestained Protein Ladder (Thermo Fisher Scientific); 2: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 3: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 4: ∼40 μg of recombinant 6xHis-tagged Vcz deaminase; 6: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 7: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 4: ∼30 μg of recombinant 6xHis-tagged URA3 deaminase.
Figure Legend Snippet: 12% SDS–PAGE gel representing the purification of recombinant Vcz and URA3 deaminases. 1, 5, and 9: PageRuler TM Prestained Protein Ladder (Thermo Fisher Scientific); 2: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 3: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 4: ∼40 μg of recombinant 6xHis-tagged Vcz deaminase; 6: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 7: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 4: ∼30 μg of recombinant 6xHis-tagged URA3 deaminase.

Techniques Used: SDS Page, Purification, Recombinant, Transformation Assay

15) Product Images from "Immunization of mice by a multimeric L2-based linear epitope (17-36) from HPV type 16/18 induced cross reactive neutralizing antibodies"

Article Title: Immunization of mice by a multimeric L2-based linear epitope (17-36) from HPV type 16/18 induced cross reactive neutralizing antibodies

Journal: Research in Pharmaceutical Sciences

doi: 10.4103/1735-5362.212043

Construction and characterization of a recombinant plasmid encoding the dual-type fusion peptide. (A) Schematic diagram of the recombinant pET28a harboring the dual-type L2 fusion peptide (pET-28a-L217-36 ×3). The synthesized dual-type L2 fragment was subcloned into the NcoI and XhoI sites of the pET-28a plasmid. (B) Gel electrophoresis of digested recombinant pET-28a-L217-36 ×3. The positive clones were confirmed using enzymatic digestion with the same enzymes. Lanes: 1, undigested plasmid; 2, DNA ladder; 3, digested plasmid.
Figure Legend Snippet: Construction and characterization of a recombinant plasmid encoding the dual-type fusion peptide. (A) Schematic diagram of the recombinant pET28a harboring the dual-type L2 fusion peptide (pET-28a-L217-36 ×3). The synthesized dual-type L2 fragment was subcloned into the NcoI and XhoI sites of the pET-28a plasmid. (B) Gel electrophoresis of digested recombinant pET-28a-L217-36 ×3. The positive clones were confirmed using enzymatic digestion with the same enzymes. Lanes: 1, undigested plasmid; 2, DNA ladder; 3, digested plasmid.

Techniques Used: Recombinant, Plasmid Preparation, Positron Emission Tomography, Synthesized, Nucleic Acid Electrophoresis, Clone Assay

16) Product Images from "Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network"

Article Title: Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network

Journal: Nature Communications

doi: 10.1038/ncomms9142

PhpCAT aromatic amino-acid transport activity. ( a ) Immunoblots on 2.5 μg of whole-cell crude extracts prepared from E. coli carrying an empty pET28a vector or codon-optimized pET28a: PhpCAT 157-1749 , and 2.5 μg of purified PhpCAT 53-583 . ( b–e ) Transport assays on whole E. coli cells carrying empty pET28a (white circles) or pET28a: PhpCAT 157-1749 (black circles). An equal number of cells were incubated with 100 μM 14 C-glucose as a negative control ( b ), 14 C-phenylalanine ( c ), 14 C-tyrosine ( d ) or 14 C-tryptophan ( e ) for the indicated time periods until termination of the assays by rapid filtration. Data are presented as a percentage relative to the corresponding empty-vector control value at 2 min set to 100%. Data are means±s.e.m. ( n ≥3 biological replicates). Two-tailed Student's t- tests revealed that relative accumulation of radiolabelled aromatic amino acids was statistically different in E. coli -expressing PhpCAT versus empty-vector control ( P ≤ 0.01).
Figure Legend Snippet: PhpCAT aromatic amino-acid transport activity. ( a ) Immunoblots on 2.5 μg of whole-cell crude extracts prepared from E. coli carrying an empty pET28a vector or codon-optimized pET28a: PhpCAT 157-1749 , and 2.5 μg of purified PhpCAT 53-583 . ( b–e ) Transport assays on whole E. coli cells carrying empty pET28a (white circles) or pET28a: PhpCAT 157-1749 (black circles). An equal number of cells were incubated with 100 μM 14 C-glucose as a negative control ( b ), 14 C-phenylalanine ( c ), 14 C-tyrosine ( d ) or 14 C-tryptophan ( e ) for the indicated time periods until termination of the assays by rapid filtration. Data are presented as a percentage relative to the corresponding empty-vector control value at 2 min set to 100%. Data are means±s.e.m. ( n ≥3 biological replicates). Two-tailed Student's t- tests revealed that relative accumulation of radiolabelled aromatic amino acids was statistically different in E. coli -expressing PhpCAT versus empty-vector control ( P ≤ 0.01).

Techniques Used: Activity Assay, Western Blot, Plasmid Preparation, Purification, Incubation, Negative Control, Filtration, Two Tailed Test, Expressing

17) Product Images from "Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil"

Article Title: Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02375

12% SDS–PAGE gel representing the purification of recombinant Vcz and URA3 deaminases. 1, 5, and 9: PageRuler TM Prestained Protein Ladder (Thermo Fisher Scientific); 2: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 3: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 4: ∼40 μg of recombinant 6xHis-tagged Vcz deaminase; 6: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 7: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 4: ∼30 μg of recombinant 6xHis-tagged URA3 deaminase.
Figure Legend Snippet: 12% SDS–PAGE gel representing the purification of recombinant Vcz and URA3 deaminases. 1, 5, and 9: PageRuler TM Prestained Protein Ladder (Thermo Fisher Scientific); 2: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 3: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET28a-Vcz; 4: ∼40 μg of recombinant 6xHis-tagged Vcz deaminase; 6: total proteins obtained from induced E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 7: soluble protein fraction of E. coli BL21(DE-3) bacteria transformed with pET21b-URA3; 4: ∼30 μg of recombinant 6xHis-tagged URA3 deaminase.

Techniques Used: SDS Page, Purification, Recombinant, Transformation Assay

18) Product Images from "ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains"

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl575

Comparison of Zα domains ( A ) and schematic representation of constructs used for transfections ( B ) and protein expression ( C ). Comparison of Zα domains of human (hs) and mouse (mm) ZBP1, human ADAR1, zebrafish (dr) PKZ, vaccinia virus (vv) E3L and yaba-like disease virus (yldv) E3L is shown (A). The structures of the mouse (mm) Zα ZBP1 , human (hs) Zα ADAR1 , yaba-like disease virus (yldv) Zα E3L domains have been determined in complex with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in other Zα domains, are boxed in light blue. Asterisks mark the conserved asparagine and tyrosine residues that have been mutated in this study in hsZBP1, as well as a conserved tryptophan. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA contacting nor structural but match the consensus sequence are highlighted in yellow. Isoleucine 335 in Zβ ADAR1 is highlighted in red. (B) The exon composition of the most prominent ZBP1 splice variants ZBP1full and ZBP1ΔZα as well as that of artificial constructs are shown. Exon 7 is rarely found in mRNA. Exon 9 contains an alternative termination site ( 22 ). ZBP1full and ZBP1ΔZα have been expressed as un-tagged or GFP tagged proteins in HeLa cells. ZBP1ΔZβ, ZBP1ΔZαΔZβ, ZBP1E1-5 and ZBP1E1-5ΔZα were expressed as GFP-tagged proteins. Schematic representation of the exon composition of constructs expressed from pET28a (p28) vectors in E.coli are shown in (C).
Figure Legend Snippet: Comparison of Zα domains ( A ) and schematic representation of constructs used for transfections ( B ) and protein expression ( C ). Comparison of Zα domains of human (hs) and mouse (mm) ZBP1, human ADAR1, zebrafish (dr) PKZ, vaccinia virus (vv) E3L and yaba-like disease virus (yldv) E3L is shown (A). The structures of the mouse (mm) Zα ZBP1 , human (hs) Zα ADAR1 , yaba-like disease virus (yldv) Zα E3L domains have been determined in complex with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in other Zα domains, are boxed in light blue. Asterisks mark the conserved asparagine and tyrosine residues that have been mutated in this study in hsZBP1, as well as a conserved tryptophan. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA contacting nor structural but match the consensus sequence are highlighted in yellow. Isoleucine 335 in Zβ ADAR1 is highlighted in red. (B) The exon composition of the most prominent ZBP1 splice variants ZBP1full and ZBP1ΔZα as well as that of artificial constructs are shown. Exon 7 is rarely found in mRNA. Exon 9 contains an alternative termination site ( 22 ). ZBP1full and ZBP1ΔZα have been expressed as un-tagged or GFP tagged proteins in HeLa cells. ZBP1ΔZβ, ZBP1ΔZαΔZβ, ZBP1E1-5 and ZBP1E1-5ΔZα were expressed as GFP-tagged proteins. Schematic representation of the exon composition of constructs expressed from pET28a (p28) vectors in E.coli are shown in (C).

Techniques Used: Construct, Transfection, Expressing, Sequencing

19) Product Images from "GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance"

Article Title: GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance

Journal: Scientific Reports

doi: 10.1038/s41598-018-23806-4

Heterologous expression of MG detoxification enzymes in E. coli provides varying tolerance to various abiotic stresses: The BL21 E. coli cells containing the constructs of MG detoxification genes (pET28a-AtGLYI, pET28a-AtGLYII and pET28a-AtD-LDH) were grown in presence of different abiotic stresses such as ( A ) salinity (200 mM NaCl), ( B ) oxidative (5 mM H 2 O 2 ) and ( C ) exogenous MG (0.5 mM MG) and their growth was monitored. Cells containing empty vector were used as control.
Figure Legend Snippet: Heterologous expression of MG detoxification enzymes in E. coli provides varying tolerance to various abiotic stresses: The BL21 E. coli cells containing the constructs of MG detoxification genes (pET28a-AtGLYI, pET28a-AtGLYII and pET28a-AtD-LDH) were grown in presence of different abiotic stresses such as ( A ) salinity (200 mM NaCl), ( B ) oxidative (5 mM H 2 O 2 ) and ( C ) exogenous MG (0.5 mM MG) and their growth was monitored. Cells containing empty vector were used as control.

Techniques Used: Expressing, Construct, Plasmid Preparation

20) Product Images from "Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein"

Article Title: Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein

Journal: Indian Journal of Virology

doi: 10.1007/s13337-013-0159-7

Expression of coat protein (CP) of Large cardamom chirke virus (LCCV) in E. coli. a SDS-PAGE b Western blot with the antiserum developed against the gel extracted recombinant CP. Lane 1 : Marker, Lane 2 : Uninduced BL21(DE3) E. coli cells harboring the pET28a-CP,
Figure Legend Snippet: Expression of coat protein (CP) of Large cardamom chirke virus (LCCV) in E. coli. a SDS-PAGE b Western blot with the antiserum developed against the gel extracted recombinant CP. Lane 1 : Marker, Lane 2 : Uninduced BL21(DE3) E. coli cells harboring the pET28a-CP,

Techniques Used: Expressing, SDS Page, Western Blot, Recombinant, Marker

21) Product Images from "GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance"

Article Title: GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance

Journal: Scientific Reports

doi: 10.1038/s41598-018-23806-4

Heterologous expression of MG detoxification enzymes in E. coli provides varying tolerance to various abiotic stresses: The BL21 E. coli cells containing the constructs of MG detoxification genes (pET28a-AtGLYI, pET28a-AtGLYII and pET28a-AtD-LDH) were grown in presence of different abiotic stresses such as ( A ) salinity (200 mM NaCl), ( B ) oxidative (5 mM H 2 O 2 ) and ( C ) exogenous MG (0.5 mM MG) and their growth was monitored. Cells containing empty vector were used as control.
Figure Legend Snippet: Heterologous expression of MG detoxification enzymes in E. coli provides varying tolerance to various abiotic stresses: The BL21 E. coli cells containing the constructs of MG detoxification genes (pET28a-AtGLYI, pET28a-AtGLYII and pET28a-AtD-LDH) were grown in presence of different abiotic stresses such as ( A ) salinity (200 mM NaCl), ( B ) oxidative (5 mM H 2 O 2 ) and ( C ) exogenous MG (0.5 mM MG) and their growth was monitored. Cells containing empty vector were used as control.

Techniques Used: Expressing, Construct, Plasmid Preparation

22) Product Images from "Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure"

Article Title: Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure

Journal: SpringerPlus

doi: 10.1186/s40064-016-2381-4

Expression and purification of the recombination PmMuGST fusion protein. Equal amounts of proteins (30 μg) were subject to SDS-PAGE and western blotting analysis. a Protein samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M , protein standard; lane 1 , crude extract of BL 21 (DE3) without plasmid; lane 2 , crude extract of the transformed BL21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 3 , purified PmMuGST fusion protein. b Protein samples were analyzed by immunoblotting with anti- PmMuGST antibody. Lane M , protein standard; lane 1 , crude extract of the transformed BL 21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 2 , purified PmMuGST fusion protein
Figure Legend Snippet: Expression and purification of the recombination PmMuGST fusion protein. Equal amounts of proteins (30 μg) were subject to SDS-PAGE and western blotting analysis. a Protein samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M , protein standard; lane 1 , crude extract of BL 21 (DE3) without plasmid; lane 2 , crude extract of the transformed BL21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 3 , purified PmMuGST fusion protein. b Protein samples were analyzed by immunoblotting with anti- PmMuGST antibody. Lane M , protein standard; lane 1 , crude extract of the transformed BL 21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 2 , purified PmMuGST fusion protein

Techniques Used: Expressing, Purification, SDS Page, Western Blot, Staining, Plasmid Preparation, Transformation Assay

23) Product Images from "Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae"

Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-12-16

Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).
Figure Legend Snippet: Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).

Techniques Used: Agarose Gel Electrophoresis, Modification, Incubation

24) Product Images from "Control of Phage Bxb1 Excision by a Novel Recombination Directionality FactorA Novel Phage Protein Mediates the Virus's Removal from Bacterial Chromosomes"

Article Title: Control of Phage Bxb1 Excision by a Novel Recombination Directionality FactorA Novel Phage Protein Mediates the Virus's Removal from Bacterial Chromosomes

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0040186

In Vitro Excisive Recombination Using gp47 (A) E. coli BL21(DE3)pLysS transformed with pET28a and pPGgp47 were grown to an A 600 of 0.6 at 30 °C and induced for an additional 4 h at 22 °C with 0.6 mM IPTG. The cells were lysed in lysis buffer (see Materials and Methods ) and partially purified by passage through a Ni-NTA column followed by elution with 150 mM imidazole; I, S, Ft, W, and E represent the insoluble fraction, soluble fraction, flow-through from the Ni-NTA column, washes with the indicated concentration of imidazole, and 150 mM elution from the Ni-NTA column, respectively. The induced cells of pPGgp47 show the presence of an approximately 32-kDa protein (as indicated) that is absent from the pET28a expression lanes and is abundant in the insoluble fraction. Molecular weight markers are shown in lane M and their corresponding sizes indicated. (B) Integrative recombination was performed as described previously using a supercoiled attP substrate, a linear 50-bp attB DNA, and increasing concentrations of gpInt [ 34 ]: panel a, in the absence of any additional protein; panel b, in the presence of partially purified gp47; panel c, with addition of a control extract. Lanes 1–5 contain 0.36, 0.18, 0.09, 0.045, and 0.0225 μM of gpInt respectively. Panels b and c contain 1.78 μM of gp47 and an equivalent amount of the control extract respectively, in addition to gpInt. The positions of the supercoiled substrate and the linear recombinant product are indicated. The small (50 bp) linear attB substrate migrates fast and is not shown. (C) Excisive recombination was carried out in recombination buffer (see Materials and Methods ) using a 367-bp attL in a supercoiled plasmid and a 377-bp linear attR partner DNA. Lanes 1–5 of panel a contain increasing concentrations of gp47 (0.89, 1.78, 2.67, 3.56, and 5.34 μM), lanes 7–11 of panel b contain an equivalent amount of the control protein. Control reactions lacking either the partner attR DNA (lane12), gp47 (lane 13), or gpInt (lane14) are shown in panel c. The positions of the supercoiled attL substrate and the linear recombinant product are indicated. (D) Excisive recombination reactions with varying sizes of linear DNA substrates show that only small substrate sites are required. Panel a shows recombination between a supercoiled plasmid containing a 367-bp attL and varying sizes of linear attR partner DNA as indicated. Panel b shows recombination between a supercoiled plasmid containing a 377-bp attR and varying sizes of linear attL partner DNAs. The positions of supercoiled substrates and linear recombinant products are indicated.
Figure Legend Snippet: In Vitro Excisive Recombination Using gp47 (A) E. coli BL21(DE3)pLysS transformed with pET28a and pPGgp47 were grown to an A 600 of 0.6 at 30 °C and induced for an additional 4 h at 22 °C with 0.6 mM IPTG. The cells were lysed in lysis buffer (see Materials and Methods ) and partially purified by passage through a Ni-NTA column followed by elution with 150 mM imidazole; I, S, Ft, W, and E represent the insoluble fraction, soluble fraction, flow-through from the Ni-NTA column, washes with the indicated concentration of imidazole, and 150 mM elution from the Ni-NTA column, respectively. The induced cells of pPGgp47 show the presence of an approximately 32-kDa protein (as indicated) that is absent from the pET28a expression lanes and is abundant in the insoluble fraction. Molecular weight markers are shown in lane M and their corresponding sizes indicated. (B) Integrative recombination was performed as described previously using a supercoiled attP substrate, a linear 50-bp attB DNA, and increasing concentrations of gpInt [ 34 ]: panel a, in the absence of any additional protein; panel b, in the presence of partially purified gp47; panel c, with addition of a control extract. Lanes 1–5 contain 0.36, 0.18, 0.09, 0.045, and 0.0225 μM of gpInt respectively. Panels b and c contain 1.78 μM of gp47 and an equivalent amount of the control extract respectively, in addition to gpInt. The positions of the supercoiled substrate and the linear recombinant product are indicated. The small (50 bp) linear attB substrate migrates fast and is not shown. (C) Excisive recombination was carried out in recombination buffer (see Materials and Methods ) using a 367-bp attL in a supercoiled plasmid and a 377-bp linear attR partner DNA. Lanes 1–5 of panel a contain increasing concentrations of gp47 (0.89, 1.78, 2.67, 3.56, and 5.34 μM), lanes 7–11 of panel b contain an equivalent amount of the control protein. Control reactions lacking either the partner attR DNA (lane12), gp47 (lane 13), or gpInt (lane14) are shown in panel c. The positions of the supercoiled attL substrate and the linear recombinant product are indicated. (D) Excisive recombination reactions with varying sizes of linear DNA substrates show that only small substrate sites are required. Panel a shows recombination between a supercoiled plasmid containing a 367-bp attL and varying sizes of linear attR partner DNA as indicated. Panel b shows recombination between a supercoiled plasmid containing a 377-bp attR and varying sizes of linear attL partner DNAs. The positions of supercoiled substrates and linear recombinant products are indicated.

Techniques Used: In Vitro, Transformation Assay, Lysis, Purification, Flow Cytometry, Concentration Assay, Expressing, Molecular Weight, Recombinant, Plasmid Preparation

25) Product Images from "Triticum aestivum WRAB18 functions in plastids and confers abiotic stress tolerance when overexpressed in Escherichia coli and Nicotiania benthamiana"

Article Title: Triticum aestivum WRAB18 functions in plastids and confers abiotic stress tolerance when overexpressed in Escherichia coli and Nicotiania benthamiana

Journal: PLoS ONE

doi: 10.1371/journal.pone.0171340

Growth curves of Escherichia coli cultures transformed with WRAB18 or control pET28a under four abiotic stresses. E . coli strains grown under standard culture conditions (A) , in medium supplemented with 800 mM mannitol (B) or 500 mM NaCl (C) , and under exposure to 28° C (D) or 45° C (E) . The OD 600 was measured as an indicator of the increase in density of the liquid cultures. Each stress assay was performed three times, and statistically significant differences were analyzed using the Student’s t-test.
Figure Legend Snippet: Growth curves of Escherichia coli cultures transformed with WRAB18 or control pET28a under four abiotic stresses. E . coli strains grown under standard culture conditions (A) , in medium supplemented with 800 mM mannitol (B) or 500 mM NaCl (C) , and under exposure to 28° C (D) or 45° C (E) . The OD 600 was measured as an indicator of the increase in density of the liquid cultures. Each stress assay was performed three times, and statistically significant differences were analyzed using the Student’s t-test.

Techniques Used: Transformation Assay

26) Product Images from "Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification"

Article Title: Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification

Journal: Scientific Reports

doi: 10.1038/srep21051

( A ) Scheme of the glycosylation reaction based on which the high-throughput screening method was developed. ( B ) The representative activity data of glycosylation of fluorescent 4-MU illustrating ~100 random members from the GTF-D saturation mutagenesis library screening. The wild-type enzyme and mutant M4 were indicated. Strain BL21(DE3) harboring plasmid pET28a was used as control. Activities were calculated as the fluorescence differences of the variants between 0 (before the reaction) and 5 h (after the reaction).
Figure Legend Snippet: ( A ) Scheme of the glycosylation reaction based on which the high-throughput screening method was developed. ( B ) The representative activity data of glycosylation of fluorescent 4-MU illustrating ~100 random members from the GTF-D saturation mutagenesis library screening. The wild-type enzyme and mutant M4 were indicated. Strain BL21(DE3) harboring plasmid pET28a was used as control. Activities were calculated as the fluorescence differences of the variants between 0 (before the reaction) and 5 h (after the reaction).

Techniques Used: High Throughput Screening Assay, Activity Assay, Mutagenesis, Library Screening, Plasmid Preparation, Fluorescence

27) Product Images from "Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli"

Article Title: Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156692

(a) Cell growth and activity of L-asparaginase using E . coli BL21 (DE3)/pET26b/ ans (extracellular expression) induced after 203 minutes of culture; (b) growth and expression of the protein using E . coli BL21 (DE3)/pET28a/ ans (intracellular expression) induced after 198 minutes of culture; (c) production of acetic acid over time; (d) specific activity of L-asparaginase over time as of induction of protein expression using the recombinant strains of L-asparaginase ( E . coli BL21 (DE3)/pET26b/ ans and E . coli BL21 (DE3)/pET28a/ ans ). The results are the mean of the data, with error bars representing the standard deviation of triplicate values.
Figure Legend Snippet: (a) Cell growth and activity of L-asparaginase using E . coli BL21 (DE3)/pET26b/ ans (extracellular expression) induced after 203 minutes of culture; (b) growth and expression of the protein using E . coli BL21 (DE3)/pET28a/ ans (intracellular expression) induced after 198 minutes of culture; (c) production of acetic acid over time; (d) specific activity of L-asparaginase over time as of induction of protein expression using the recombinant strains of L-asparaginase ( E . coli BL21 (DE3)/pET26b/ ans and E . coli BL21 (DE3)/pET28a/ ans ). The results are the mean of the data, with error bars representing the standard deviation of triplicate values.

Techniques Used: Activity Assay, Expressing, Recombinant, Standard Deviation

28) Product Images from "Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor"

Article Title: Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019131

Cloning, expression and functional analysis of M. tuberculosis mfd (Mtbmfd) . A . Schematic showing the strategy used for cloning of Mtbmfd ; F1, F2 F3 represents three fragments of Mtbmfd obtained by PCR amplification from genomic DNA of M. tuberculosis by using set of specific primers. F1 was cloned into pET32a in MscI-KpnI sites and F2 was cloned into pGPS3 vector using KpnI-BsrB1 sites. F2+F3 fragment was obtained by ligation of F3 into PGPS3 containing F2 using BsrBI-HindIII sites. A 2.4 kb fragment containing F2+F3 was released using KpnI-HindIII sites from pGPS3 clone and ligated into pET32a F1 clone to obtain full length Mtb mfd gene. Sub cloning of Mtb mfd in pET28a and pTrc99A vectors were used for overexpression and in vivo assays respectively. B . SDS-PAGE analysis of overexpression of His-tagged MtbMfd in E. coli expression strain (Tuner) using pETmfd construct. Lane 1, total cell extracts of Tuner cells; lane 2, total cell extracts of uninduced Tuner cells carrying pET28a vector alone; lane 3, induced cell extracts of Tuner cells harboring pET28a vector; lane 4, protein molecular weight marker; lane 5, total cell extracts of uninduced Tuner cells carrying pETmfd construct and lane 6, total cell extract of induced cell extracts (0.3 mM IPTG) of Tuner cells carrying pETmfd construct. C . Western blot analysis using anti-MtbMfd polyclonal antibody for expression of Mfd in E. coli stains used for complementation studies; lane 1, AB1157; lane 2, UNCNOMFD; lane 3, pTrcmfd in presence of 0.5 mM of IPTG and lane 4, pTrcmfd. D . Effect of UV on survival (S/S 0 ) of E. coli strains; AB1157 (red, ▪); UNCNOMFD (blue, ▴); pTrcmfd (brown,▾) and pTrc99A (black,♦). Each value represents the average from three independent experiments. (Survival = S/S 0 ; where S 0 = number of bacterial colonies obtained without UV irradiation and S = number of bacterial colonies obtained after UV irradiation). (AB1157, E. coli wild-type strain; UNCNOMFD, mfd deficient stain of E. coli ; pTrcmfd, UNCNOMFD transformed with Mtb mfd construct and pTrc99A, UNCNOMFD transformed with pTrc99A vector alone).
Figure Legend Snippet: Cloning, expression and functional analysis of M. tuberculosis mfd (Mtbmfd) . A . Schematic showing the strategy used for cloning of Mtbmfd ; F1, F2 F3 represents three fragments of Mtbmfd obtained by PCR amplification from genomic DNA of M. tuberculosis by using set of specific primers. F1 was cloned into pET32a in MscI-KpnI sites and F2 was cloned into pGPS3 vector using KpnI-BsrB1 sites. F2+F3 fragment was obtained by ligation of F3 into PGPS3 containing F2 using BsrBI-HindIII sites. A 2.4 kb fragment containing F2+F3 was released using KpnI-HindIII sites from pGPS3 clone and ligated into pET32a F1 clone to obtain full length Mtb mfd gene. Sub cloning of Mtb mfd in pET28a and pTrc99A vectors were used for overexpression and in vivo assays respectively. B . SDS-PAGE analysis of overexpression of His-tagged MtbMfd in E. coli expression strain (Tuner) using pETmfd construct. Lane 1, total cell extracts of Tuner cells; lane 2, total cell extracts of uninduced Tuner cells carrying pET28a vector alone; lane 3, induced cell extracts of Tuner cells harboring pET28a vector; lane 4, protein molecular weight marker; lane 5, total cell extracts of uninduced Tuner cells carrying pETmfd construct and lane 6, total cell extract of induced cell extracts (0.3 mM IPTG) of Tuner cells carrying pETmfd construct. C . Western blot analysis using anti-MtbMfd polyclonal antibody for expression of Mfd in E. coli stains used for complementation studies; lane 1, AB1157; lane 2, UNCNOMFD; lane 3, pTrcmfd in presence of 0.5 mM of IPTG and lane 4, pTrcmfd. D . Effect of UV on survival (S/S 0 ) of E. coli strains; AB1157 (red, ▪); UNCNOMFD (blue, ▴); pTrcmfd (brown,▾) and pTrc99A (black,♦). Each value represents the average from three independent experiments. (Survival = S/S 0 ; where S 0 = number of bacterial colonies obtained without UV irradiation and S = number of bacterial colonies obtained after UV irradiation). (AB1157, E. coli wild-type strain; UNCNOMFD, mfd deficient stain of E. coli ; pTrcmfd, UNCNOMFD transformed with Mtb mfd construct and pTrc99A, UNCNOMFD transformed with pTrc99A vector alone).

Techniques Used: Clone Assay, Expressing, Functional Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Ligation, Subcloning, Over Expression, In Vivo, SDS Page, Construct, Molecular Weight, Marker, Western Blot, Irradiation, Staining, Transformation Assay

29) Product Images from "Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications"

Article Title: Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-12-27

Schematic diagram of the constructs used for TAT-Apoptin protein expression. ( A ) Schematic representation of the TAT-Apoptin protein fused with different affinity tags together with the expression vectors used in this study. The designations of the TAT-Apoptin protein and its expression vectors are indicated, ( a ), ( b ), ( c ) and ( d ). The constructs, ( a ) and ( b ), contain the full-length TAT-VP3 gene cloned into the vectors pET28a and pGEX-4 T-1; these were used for expression of TAT-Apoptin protein with either a six-histidine (6 × His) tag or a glutathione-s-transferase (GST) tag at the N-terminus, respectively. Constructs ( c ) and ( d ) containing the TAT-VP3 gene that was codon-optimized; this was derived from construct ( b ) by replacing rare codons without altering the amino acid sequence. The codon-optimized TAT-VP3gene, TAT-VP3 opt , was then cloned into pET28a and pGEX-4 T-1. ( B ) Sequence comparison between the TAT-VP3 gene and the TAT-VP3 opt gene. The nucleotide sequences were compared between the original TAT-VP3 gene (wild type TAT-VP3) and the sequence of codon-optimized TAT-VP3 gene (TAT-VP3 Opt ) over the whole coding region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: Schematic diagram of the constructs used for TAT-Apoptin protein expression. ( A ) Schematic representation of the TAT-Apoptin protein fused with different affinity tags together with the expression vectors used in this study. The designations of the TAT-Apoptin protein and its expression vectors are indicated, ( a ), ( b ), ( c ) and ( d ). The constructs, ( a ) and ( b ), contain the full-length TAT-VP3 gene cloned into the vectors pET28a and pGEX-4 T-1; these were used for expression of TAT-Apoptin protein with either a six-histidine (6 × His) tag or a glutathione-s-transferase (GST) tag at the N-terminus, respectively. Constructs ( c ) and ( d ) containing the TAT-VP3 gene that was codon-optimized; this was derived from construct ( b ) by replacing rare codons without altering the amino acid sequence. The codon-optimized TAT-VP3gene, TAT-VP3 opt , was then cloned into pET28a and pGEX-4 T-1. ( B ) Sequence comparison between the TAT-VP3 gene and the TAT-VP3 opt gene. The nucleotide sequences were compared between the original TAT-VP3 gene (wild type TAT-VP3) and the sequence of codon-optimized TAT-VP3 gene (TAT-VP3 Opt ) over the whole coding region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.

Techniques Used: Construct, Expressing, Clone Assay, Derivative Assay, Sequencing

30) Product Images from "Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)"

Article Title: Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0155096

SDS-PAGE analysis of rGmolGOBP1 (A) and rGmolGOBP2 (B) in Escherichia coli . M. Protein molecular weight marker; 1. Non-induced pET28a(+)/GmolGOBP1transformed BL21 (DE3) cells; 2. Induced pET28a(+)/GmolGOBP1transformed BL21 (DE3) cells; 3. Supernatant of disrupted pET28a(+)/GmolGOBP1 IPTG induced cells; 4. Inclusion body of disrupted pET28a(+)/GmolGOBP1 IPTG induced cells; 5. Purified rGmolGOBP1; 6. Non-induced pET28a (+)/GmolGOBP2 transformed BL21 (DE3) cells; 7. Induced pET28a(+)/GmolGOBP2 transformed BL21 (DE3) cells; 8. Supernatant of disrupted pET28a(+)/GmolGOBP2 IPTG induced cells; 9. Inclusion body of induced pET28a(+)/GmolGOBP2 IPTG induced cells; 10. Purified rGmolGOBP2.
Figure Legend Snippet: SDS-PAGE analysis of rGmolGOBP1 (A) and rGmolGOBP2 (B) in Escherichia coli . M. Protein molecular weight marker; 1. Non-induced pET28a(+)/GmolGOBP1transformed BL21 (DE3) cells; 2. Induced pET28a(+)/GmolGOBP1transformed BL21 (DE3) cells; 3. Supernatant of disrupted pET28a(+)/GmolGOBP1 IPTG induced cells; 4. Inclusion body of disrupted pET28a(+)/GmolGOBP1 IPTG induced cells; 5. Purified rGmolGOBP1; 6. Non-induced pET28a (+)/GmolGOBP2 transformed BL21 (DE3) cells; 7. Induced pET28a(+)/GmolGOBP2 transformed BL21 (DE3) cells; 8. Supernatant of disrupted pET28a(+)/GmolGOBP2 IPTG induced cells; 9. Inclusion body of induced pET28a(+)/GmolGOBP2 IPTG induced cells; 10. Purified rGmolGOBP2.

Techniques Used: SDS Page, Molecular Weight, Marker, Purification, Transformation Assay

31) Product Images from "A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB"

Article Title: A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB

Journal: Protein Expression and Purification

doi: 10.1016/j.pep.2017.01.010

The pBDDP-SPR3 expression vector . (A) General structure of the recombinant protein produced from pBDDP-SPR3 showing two N-terminal His 8 tags separated by a 28 amino acid linker followed by a TEV cleavage site. (B) The sequence and features of the pBDDP-SPR3 tag and multiple cloning site: This cassette was designed with flanking NcoI and XhoI sites to allow cloning into pET28a. This resulted in the removal of the entire pET28a tag and MCS region and replacement with the BDDP-SPR3 insert.
Figure Legend Snippet: The pBDDP-SPR3 expression vector . (A) General structure of the recombinant protein produced from pBDDP-SPR3 showing two N-terminal His 8 tags separated by a 28 amino acid linker followed by a TEV cleavage site. (B) The sequence and features of the pBDDP-SPR3 tag and multiple cloning site: This cassette was designed with flanking NcoI and XhoI sites to allow cloning into pET28a. This resulted in the removal of the entire pET28a tag and MCS region and replacement with the BDDP-SPR3 insert.

Techniques Used: Expressing, Plasmid Preparation, Recombinant, Produced, Sequencing, Clone Assay

32) Product Images from "Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody"

Article Title: Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody

Journal: Iranian Red Crescent Medical Journal

doi: 10.5812/ircmj.28271

Screening of the fliC Gene by Restriction Enzyme Digestion The plasmids were extracted and digested with the appropriate restriction enzymes. Lane 1, pET-28a digested with BamHI ; lane 2, pET28a/ fliC digested with BamHI ; lane 3, pET28a/ fliC digested with BamHI and HindIII ; and lane M, 1 kb DNA size marker. The products were electrophoresed on 1% w/v agarose gel.
Figure Legend Snippet: Screening of the fliC Gene by Restriction Enzyme Digestion The plasmids were extracted and digested with the appropriate restriction enzymes. Lane 1, pET-28a digested with BamHI ; lane 2, pET28a/ fliC digested with BamHI ; lane 3, pET28a/ fliC digested with BamHI and HindIII ; and lane M, 1 kb DNA size marker. The products were electrophoresed on 1% w/v agarose gel.

Techniques Used: Positron Emission Tomography, Marker, Agarose Gel Electrophoresis

33) Product Images from "Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis"

Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr660

Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.
Figure Legend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

Techniques Used: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

34) Product Images from "The restriction endonuclease R.NmeDI from Neisseria meningitidis that recognizes a palindromic sequence and cuts the DNA on both sides of the recognition sequence"

Article Title: The restriction endonuclease R.NmeDI from Neisseria meningitidis that recognizes a palindromic sequence and cuts the DNA on both sides of the recognition sequence

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm702

Determination of the recognition sequence of R.NmeDI. ( A ) Comparison of the plasmid DNA cleavage by R.NmeDI and R.Cfr10I. Lane 1, pBluescript KS II (+) cleaved with R.Cfr10I; lane 2, pBluescript KS II (+) cleaved with R.NmeDI; lane 3, pBADHisA cleaved with R.NmeDI; lane 4, pBADHisA cleaved with R.Cfr10I. ( B ) Double digestion of the plasmid DNA with R.Cfr10I and R.NmeDI does not change the banding profile. Lane 1, pBR322 cleaved with R.Cfr10I and R.NmeDI; lane 2, pBR322 cleaved with R.NmeDI; lane 3, pET28a cleaved with R.NmeDI; lane 4, pET28a cleaved with R.Cfr10I and R.NmeDI. When double digestion was carried out the DNA was first digested with R.Cfr10I for 180 min and then with R.NmeDI for 180 min. M, DNA molecular weight marker: 10 000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp. ( C ) R.NmeDI cleaves double-stranded DNA on both sides of its recognition sequence. Lane M, DNA Ladder, Ultra Low Range: 10, 15, 20, 25, 35, 50, 75, 100 and 150 bp; lane 1, pBluescript KS II (+) DNA digested with R.NmeDI and electrophoresed on 20% polyacrylamide gel in TBE buffer.
Figure Legend Snippet: Determination of the recognition sequence of R.NmeDI. ( A ) Comparison of the plasmid DNA cleavage by R.NmeDI and R.Cfr10I. Lane 1, pBluescript KS II (+) cleaved with R.Cfr10I; lane 2, pBluescript KS II (+) cleaved with R.NmeDI; lane 3, pBADHisA cleaved with R.NmeDI; lane 4, pBADHisA cleaved with R.Cfr10I. ( B ) Double digestion of the plasmid DNA with R.Cfr10I and R.NmeDI does not change the banding profile. Lane 1, pBR322 cleaved with R.Cfr10I and R.NmeDI; lane 2, pBR322 cleaved with R.NmeDI; lane 3, pET28a cleaved with R.NmeDI; lane 4, pET28a cleaved with R.Cfr10I and R.NmeDI. When double digestion was carried out the DNA was first digested with R.Cfr10I for 180 min and then with R.NmeDI for 180 min. M, DNA molecular weight marker: 10 000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp. ( C ) R.NmeDI cleaves double-stranded DNA on both sides of its recognition sequence. Lane M, DNA Ladder, Ultra Low Range: 10, 15, 20, 25, 35, 50, 75, 100 and 150 bp; lane 1, pBluescript KS II (+) DNA digested with R.NmeDI and electrophoresed on 20% polyacrylamide gel in TBE buffer.

Techniques Used: Sequencing, Plasmid Preparation, Molecular Weight, Marker

35) Product Images from "Role of Predicted Transmembrane Domains for Type III Translocation, Pore Formation, and Signaling by the Yersinia pseudotuberculosis YopB Protein "

Article Title: Role of Predicted Transmembrane Domains for Type III Translocation, Pore Formation, and Signaling by the Yersinia pseudotuberculosis YopB Protein

Journal: Infection and Immunity

doi: 10.1128/IAI.73.4.2433-2443.2005

Analysis of YopB proteins containing double consecutive proline substitutions in transmembrane domain 1 or 2. (A) Immunoblot analysis of YopB, YopBTM1, and YopBTM2 secreted by Y. pseudotuberculosis under low-calcium conditions. YP40/pYopB (B40/B + ; lane 1), YP40 (B40; lane 2), YP40/pYopBTM1 (B40/BTM1; lane 3), and YP40/pYopBTM2 (B40/BTM2; lane 4) were grown under low-calcium conditions in the absence of IPTG. Cultures were centrifuged, and proteins secreted into the supernatants were analyzed by anti-YopB immunoblotting. (B) Immunoblot analysis of YopB, YopBTM1, and YopBTM2 expressed in E. coli in the presence or absence of SycD. TUNER(DE3) pLacI cells transformed with pYopB (B + ), pYopBTM1 (BTM1), or pYopBTM2 (BTM2) alone or with pET28a-SycD (+SycD) were grown to log phase, induced with IPTG, and lysed with detergent. Samples of total bacterial cell lysate (lanes 1, 2, 5, 6, 9, and 10) or soluble fractions (lanes 3, 4, 7, 8, 11, and 12) were analyzed by immunoblotting with anti-YopB antibody.
Figure Legend Snippet: Analysis of YopB proteins containing double consecutive proline substitutions in transmembrane domain 1 or 2. (A) Immunoblot analysis of YopB, YopBTM1, and YopBTM2 secreted by Y. pseudotuberculosis under low-calcium conditions. YP40/pYopB (B40/B + ; lane 1), YP40 (B40; lane 2), YP40/pYopBTM1 (B40/BTM1; lane 3), and YP40/pYopBTM2 (B40/BTM2; lane 4) were grown under low-calcium conditions in the absence of IPTG. Cultures were centrifuged, and proteins secreted into the supernatants were analyzed by anti-YopB immunoblotting. (B) Immunoblot analysis of YopB, YopBTM1, and YopBTM2 expressed in E. coli in the presence or absence of SycD. TUNER(DE3) pLacI cells transformed with pYopB (B + ), pYopBTM1 (BTM1), or pYopBTM2 (BTM2) alone or with pET28a-SycD (+SycD) were grown to log phase, induced with IPTG, and lysed with detergent. Samples of total bacterial cell lysate (lanes 1, 2, 5, 6, 9, and 10) or soluble fractions (lanes 3, 4, 7, 8, 11, and 12) were analyzed by immunoblotting with anti-YopB antibody.

Techniques Used: Transformation Assay

36) Product Images from "High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression"

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression

Journal: BMC Veterinary Research

doi: 10.1186/1746-6148-10-115

A schematic diagram of the constructions used in this study and the alignment results for the expressed PiCV capsid gene. (A) The full-length wild-type and codon-optimized PiCV capsid protein genes were cloned independently into three expression vectors pET28a, pGEX-4T-1 or pET32a. The PiCV capsid protein with the various different fusion tags, namely a six-histidine (6xHis), a Glutathione-S-transferase (GST) and a Thioredoxin-coupled six-histidine (Trx) at its N-terminus were expressed by T7 or Tac promoter-driving after IPTG induction. (B) The nucleotide sequences were compared between the wild-type (WT) and the codon-optimized (OPT) PiCV capsid protein genes. The asterisk (*) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: A schematic diagram of the constructions used in this study and the alignment results for the expressed PiCV capsid gene. (A) The full-length wild-type and codon-optimized PiCV capsid protein genes were cloned independently into three expression vectors pET28a, pGEX-4T-1 or pET32a. The PiCV capsid protein with the various different fusion tags, namely a six-histidine (6xHis), a Glutathione-S-transferase (GST) and a Thioredoxin-coupled six-histidine (Trx) at its N-terminus were expressed by T7 or Tac promoter-driving after IPTG induction. (B) The nucleotide sequences were compared between the wild-type (WT) and the codon-optimized (OPT) PiCV capsid protein genes. The asterisk (*) represents the fact that the aligned nucleotides are identical.

Techniques Used: Clone Assay, Expressing

37) Product Images from "Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration"

Article Title: Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006980

SDS-PAGE analysis of different samples taken during the purification process of esterase LipC. Lane 1: crude supernatant of pET28a-LipC non-induced culture; Lanes 2 and 6: crude extract of induced culture; Lane 3: purification on Ni-NTA affinity Sepharose column; Lane 4: further purification with DEAE column; Lanes 5 and 6: further purification with Sephadex-G200. Lanes 1 to 5 were stained with Coomassie Brilliant Blue and Lanes 6 was stained with α-naphtyl acetate and Fast Blue for the detection of hydrolase activity.
Figure Legend Snippet: SDS-PAGE analysis of different samples taken during the purification process of esterase LipC. Lane 1: crude supernatant of pET28a-LipC non-induced culture; Lanes 2 and 6: crude extract of induced culture; Lane 3: purification on Ni-NTA affinity Sepharose column; Lane 4: further purification with DEAE column; Lanes 5 and 6: further purification with Sephadex-G200. Lanes 1 to 5 were stained with Coomassie Brilliant Blue and Lanes 6 was stained with α-naphtyl acetate and Fast Blue for the detection of hydrolase activity.

Techniques Used: SDS Page, Purification, Staining, Activity Assay

38) Product Images from "Recombination and identification of human alpha B-crystallin"

Article Title: Recombination and identification of human alpha B-crystallin

Journal: International Journal of Ophthalmology

doi: 10.18240/ijo.2018.12.06

Gel electrophoresis following Ecor l and XhoI double enzymatic digestion A: Recombinant plasmid PMD19-T-αB-crystallin; B: Recombinant plasmid pET28a-αB-crystallin. Lane 1: Marker; Lane 2: Target gene fragment.
Figure Legend Snippet: Gel electrophoresis following Ecor l and XhoI double enzymatic digestion A: Recombinant plasmid PMD19-T-αB-crystallin; B: Recombinant plasmid pET28a-αB-crystallin. Lane 1: Marker; Lane 2: Target gene fragment.

Techniques Used: Nucleic Acid Electrophoresis, Recombinant, Plasmid Preparation, Marker

39) Product Images from "Characterization of the Terminal Activation Step Catalyzed by Oxygenase CmmOIV of the Chromomycin Biosynthetic Pathway from Streptomyces griseus †"

Article Title: Characterization of the Terminal Activation Step Catalyzed by Oxygenase CmmOIV of the Chromomycin Biosynthetic Pathway from Streptomyces griseus †

Journal: Biochemistry

doi: 10.1021/bi1016205

Cloning of the cmmOIV Gene in pET28a
Figure Legend Snippet: Cloning of the cmmOIV Gene in pET28a

Techniques Used: Clone Assay

40) Product Images from "The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus"

Article Title: The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Overproduction and purification of RheA. SDS/PAGE analysis of crude extracts from E. coli BL21λDE3 carrying pET28a (lane 2) or pOLP12B (lane 3) and purified RheA protein after Ni-NTA affinity chromatography (lane 4). Molecular mass standards (New England Biolabs) were loaded in lane 1.
Figure Legend Snippet: Overproduction and purification of RheA. SDS/PAGE analysis of crude extracts from E. coli BL21λDE3 carrying pET28a (lane 2) or pOLP12B (lane 3) and purified RheA protein after Ni-NTA affinity chromatography (lane 4). Molecular mass standards (New England Biolabs) were loaded in lane 1.

Techniques Used: Purification, SDS Page, Affinity Chromatography

41) Product Images from "Endogenous cellulases in animals: Isolation of ?-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes"

Article Title: Endogenous cellulases in animals: Isolation of ?-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Detection of recombinant nematode EGases in E. coli lysates on Western blot ( A ) and CMC hydrolysis assays ( B ). ( A ). Lanes: 1, HG-ENG-1 produced from pET28c; 2, pET28c control; 3, HG-ENG-2 produced from pET28a; 4, pET28a control; 5, GR-ENG-1 from vector pMAL-c2; 6, GR-ENG-2 from vector pMAL-c2; 7, pMAL-c2 control. ( B ) Detection of CMCase activity (halo) in affinity-purified heterologous cyst nematode EGases that correspond to lanes above.
Figure Legend Snippet: Detection of recombinant nematode EGases in E. coli lysates on Western blot ( A ) and CMC hydrolysis assays ( B ). ( A ). Lanes: 1, HG-ENG-1 produced from pET28c; 2, pET28c control; 3, HG-ENG-2 produced from pET28a; 4, pET28a control; 5, GR-ENG-1 from vector pMAL-c2; 6, GR-ENG-2 from vector pMAL-c2; 7, pMAL-c2 control. ( B ) Detection of CMCase activity (halo) in affinity-purified heterologous cyst nematode EGases that correspond to lanes above.

Techniques Used: Recombinant, Western Blot, Produced, Plasmid Preparation, Activity Assay, Affinity Purification

42) Product Images from "Resilin-Based Hybrid Hydrogels for Cardiovascular Tissue Engineering"

Article Title: Resilin-Based Hybrid Hydrogels for Cardiovascular Tissue Engineering

Journal: Macromolecules

doi: 10.1002/macp.201200412

(A) Agarose gel showing the BamHI/HindIII digestion of pET28aRLP48, pET28aRLP36, pET28aRLP24, pET28aRLP12, pET28a (left to right). The linearized pET28a plasmid is clearly resolved between 5000 and 6000 bp while the RLP genes are represented
Figure Legend Snippet: (A) Agarose gel showing the BamHI/HindIII digestion of pET28aRLP48, pET28aRLP36, pET28aRLP24, pET28aRLP12, pET28a (left to right). The linearized pET28a plasmid is clearly resolved between 5000 and 6000 bp while the RLP genes are represented

Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation

43) Product Images from "Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease"

Article Title: Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease

Journal: Genes & Development

doi: 10.1101/gad.932201

Expression and purification of the Mms4–Mus81 complex. ( A ) MMS4 and MUS81 were subcloned downstream of the T7 promoters of pET28a and pET11a, respectively, and further combined to yield a single plasmid, pNJ6220, carrying the indicated expression cassettes. ( B ) An extract from induced E. coli cells carrying plasmid pNJ6220 was applied to a phosphocellulose column, washed, and eluted with the indicated salt gradient. Column fractions were then resolved by SDS-PAGE, Western blotted, and probed with antiserum against Mms4 or Mus81 as indicated at right . (L) Load; (FT) flow through. ( C ) Fractions containing immunoreactive material in B were pooled, applied to a Ni-agarose column, washed, and eluted with buffer containing the indicated concentrations of imidazole. Fractions were Western blotted and probed with the indicated anti-serum. ( D ) Fractions in C were resolved by SDS-PAGE and stained with silver. (M) Molecular weight markers.
Figure Legend Snippet: Expression and purification of the Mms4–Mus81 complex. ( A ) MMS4 and MUS81 were subcloned downstream of the T7 promoters of pET28a and pET11a, respectively, and further combined to yield a single plasmid, pNJ6220, carrying the indicated expression cassettes. ( B ) An extract from induced E. coli cells carrying plasmid pNJ6220 was applied to a phosphocellulose column, washed, and eluted with the indicated salt gradient. Column fractions were then resolved by SDS-PAGE, Western blotted, and probed with antiserum against Mms4 or Mus81 as indicated at right . (L) Load; (FT) flow through. ( C ) Fractions containing immunoreactive material in B were pooled, applied to a Ni-agarose column, washed, and eluted with buffer containing the indicated concentrations of imidazole. Fractions were Western blotted and probed with the indicated anti-serum. ( D ) Fractions in C were resolved by SDS-PAGE and stained with silver. (M) Molecular weight markers.

Techniques Used: Expressing, Purification, Plasmid Preparation, SDS Page, Western Blot, Flow Cytometry, Staining, Molecular Weight

44) Product Images from "A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya"

Article Title: A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya

Journal: RNA

doi: 10.1261/rna.5230603

In vitro activity of the wild-type (D80D) and two mutant TruD synthases (D80N, D80T). ( A ) 3 H release activity of wild-type and mutant TruD. A 5-[ 3 H]uracil-containing tRNA Glu transcript was prepared and pseudouridine formation measured as release of 3 H as described in Materials and Methods. Substrate concentration was 100 nM in a reaction volume of 0.1 mL. Wild-type and mutant recombinant synthases were overexpressed from pET28a plasmids and purified (see Materials and Methods). A total of 0.05 μg of D80D (♦), no enzyme (⋄), and 0.54 μg of D80N and D80T (triangles and circles, respectively) were added. A total of 0.15 μg of wild-type or mutant enzyme was added to the reaction at 120 min (arrow). ( B ) Ψ sequence analysis of the site of in vitro Ψ formation on tRNA Glu . RNAs that had been reacted with the recombinant wild-type and mutant enzyme to completion (200 min) were obtained by phenol extraction of the mixture and analyzed by the sequencing technique described in Materials and Methods. RNA was reacted with (+) or without (−) CMC following the standard sequencing protocol. (A, G, C, U) RNA sequencing lanes. The arrow shows the position of the stop one residue 3′ to Ψ13.
Figure Legend Snippet: In vitro activity of the wild-type (D80D) and two mutant TruD synthases (D80N, D80T). ( A ) 3 H release activity of wild-type and mutant TruD. A 5-[ 3 H]uracil-containing tRNA Glu transcript was prepared and pseudouridine formation measured as release of 3 H as described in Materials and Methods. Substrate concentration was 100 nM in a reaction volume of 0.1 mL. Wild-type and mutant recombinant synthases were overexpressed from pET28a plasmids and purified (see Materials and Methods). A total of 0.05 μg of D80D (♦), no enzyme (⋄), and 0.54 μg of D80N and D80T (triangles and circles, respectively) were added. A total of 0.15 μg of wild-type or mutant enzyme was added to the reaction at 120 min (arrow). ( B ) Ψ sequence analysis of the site of in vitro Ψ formation on tRNA Glu . RNAs that had been reacted with the recombinant wild-type and mutant enzyme to completion (200 min) were obtained by phenol extraction of the mixture and analyzed by the sequencing technique described in Materials and Methods. RNA was reacted with (+) or without (−) CMC following the standard sequencing protocol. (A, G, C, U) RNA sequencing lanes. The arrow shows the position of the stop one residue 3′ to Ψ13.

Techniques Used: In Vitro, Activity Assay, Mutagenesis, Concentration Assay, Recombinant, Purification, Sequencing, RNA Sequencing Assay

Related Articles

Clone Assay:

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: .. Plasmid construction A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively. .. And the PCP gene was cloned into the pET28a-SUMO (Novagen), resulting in a PCP -containing-plasmid pYY0098.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The DNA sequence was determined from three clones and showed an open reading frame of 1863 bp.

Article Title: Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1
Article Snippet: .. For expression cloning, pET28a (Novagen, Madison, WI) served as the expression vector. .. Native 2H1NA decarboxylase was purified from crude cell extract of BC1 cells grown for 16 h at 28°C in 4 liters of mineral salt medium (MSM) ( ) containing 0.5 g/liter 2H1NA.

Centrifugation:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Seventy-two hours after infection, cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS) supplemented with phenylmethylsulfonyl fluoride (PMSF) (1 mM), and then resuspended in 10 mM Tris-HCl, pH 8, with 1 mM PMSF and sonicated. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Amplification:

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The amplicon with the desired band size was purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pGEMT (Promega, USA), as per the manufacturer’s instructions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: PCR amplification with gene-specific primers was used to confirm the presence of the gene in the virus. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The purified amplicon was ligated into a pGEMT-Easy vector (Promega, USA) per manufacturer’s instructions, followed by transformation into competent E. coli DH5α. .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: Plasmid construction A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: .. NUDIX enzyme production Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: Production and purification of ACPs/PCP The three ACP genes were amplified by PCR using relevant primers from genomic DNA of S. tsukubaensis L19. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: Total Acas RNA was isolated using the Qiagen RNeasy Mini Kit and reverse transcribed with SuperScript III First-Strand Synthesis System (Invitrogen, Paisley, UK), using primers AcRegAF and AcRegAR, that contained NheI and EcoRI sites respectively, followed by cDNA amplification with Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA). .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Synthesized:

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: .. NUDIX enzyme production Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. Subcloning of α-Luffin Gene Fragment After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Construct:

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: Plasmid construction A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. This construct carries the VP1 gene flanked by the N-terminal glutathione-S-transferase (GST) tag and will express a GST-VP1 fusion protein.

Article Title: Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1
Article Snippet: Recombinant constructs in Escherichia coli [XL1-Blue and BL21(DE3)] were routinely grown and maintained in Luria-Bertani (LB) broth (per liter) containing 10 g of Bacto tryptone, 5 g of yeast extract, and 10 g of NaCl (pH 7.2) or on LB solid medium (1.8% [wt/vol] agar) at 37°C. .. For expression cloning, pET28a (Novagen, Madison, WI) served as the expression vector.

Electrophoresis:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Electrophoresis was carried out in prepoured discontinuous 8 and 14% acrylamide-Tris-glycine gels (Novex, San Diego, Calif.). .. T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

Incubation:

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The culture was then diluted 1:40 in LB, incubated for 2 h at 30 °C and supplemented with 1 mM IPTG.

Expressing:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: Paragraph title: Construction of recombinant burI expression plasmids ... A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: .. Expression of Recombinant VbDFR in E. coli and Purification A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. ..

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site. .. Restriction mapping and sequencing were performed to confirm correct insertion.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Paragraph title: Construction of Recombinant cneI Expression Plasmids ... This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Paragraph title: Construction of Recombinant AciI Expression Plasmids ... The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: .. NUDIX enzyme production Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: Paragraph title: Cloning and expression of Acas RegA ... The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. Subcloning of α-Luffin Gene Fragment After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1
Article Snippet: .. For expression cloning, pET28a (Novagen, Madison, WI) served as the expression vector. .. Native 2H1NA decarboxylase was purified from crude cell extract of BC1 cells grown for 16 h at 28°C in 4 liters of mineral salt medium (MSM) ( ) containing 0.5 g/liter 2H1NA.

Western Blot:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Paragraph title: SDS-PAGE and Western blotting. ... T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: The cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Transformation Assay:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The resulting recombinant plasmid (designated pGEMT-burI ) was transformed into E. coli JM109 ( ). .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The purified amplicon was ligated into a pGEMT-Easy vector (Promega, USA) per manufacturer’s instructions, followed by transformation into competent E. coli DH5α. .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The resulting recombinant plasmid (designated pDrive-aciI) was transformed into E. coli DH5α ( ). .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. To obtain Acas RegA protein, plasmid pET-AcRegA was transformed into E.coli BL21DE3.

Derivative Assay:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: .. T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen. ..

Gel Purification:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

Infection:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Seventy-two hours after infection, cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS) supplemented with phenylmethylsulfonyl fluoride (PMSF) (1 mM), and then resuspended in 10 mM Tris-HCl, pH 8, with 1 mM PMSF and sonicated. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Generated:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Anti-peptide antisera to a peptide specific for MSG 32 (KMYGLFYGSGKEWFKKLLEKIM, corresponding to amino acids 461 to 482) (see Fig. ) and to a conserved human P. carinii MSG epitope contained within the recombinant carboxyl-terminal fragment (TITSTITSKITLTST, corresponding to amino acids 968 to 982 of MSG 32) were commercially generated in rabbits by the multiple antigenic peptide method ( ) (Research Genetics, Huntsville, Ala.). .. T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

DNA Sequencing:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. The sequence of aciI cloned into pDrive and pET28a plasmids was verified by automated Sanger DNA sequencing.

Polymerase Chain Reaction:

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR product was verified using agarose gel electrophoresis followed by ethidium bromide (Sigma, St. Louis, MO) staining. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: Expression of Recombinant VbDFR in E. coli and Purification A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. .. Approximately 1.0 ng of the T-VbDFR plasmid was used as template for PCR, which was carried out using Ex-taq DNA polymerase (Takara, Japan) as described above.

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site. .. Restriction mapping and sequencing were performed to confirm correct insertion.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Upon running agarose gel electrophoresis, the desired amplicon was extracted using Wizard® SV Gel and PCR Clean-Up System (Promega, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: Plasmid construction A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. To generate the plasmid harboring the codon optimized nucleotide sequence, a codon optimized fragment encoding the first 107 amino acid residues of the N-terminus of the VP1 capsid protein was fused with the C-terminus of the VP1 protein using the 5' and 3' ends of above two DNA fragments and overlapping PCR [ ].

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: The resultant products were cloned into pTA2 vector (Toyobo) directly and sequenced to confirm PCR fidelity. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: To identify intron positions, we amplified a cDNA from Acas mRNA by reverse transcripion PCR. .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Sonication:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Seventy-two hours after infection, cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS) supplemented with phenylmethylsulfonyl fluoride (PMSF) (1 mM), and then resuspended in 10 mM Tris-HCl, pH 8, with 1 mM PMSF and sonicated. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Recombinant:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Anti-peptide antisera to a peptide specific for MSG 32 (KMYGLFYGSGKEWFKKLLEKIM, corresponding to amino acids 461 to 482) (see Fig. ) and to a conserved human P. carinii MSG epitope contained within the recombinant carboxyl-terminal fragment (TITSTITSKITLTST, corresponding to amino acids 968 to 982 of MSG 32) were commercially generated in rabbits by the multiple antigenic peptide method ( ) (Research Genetics, Huntsville, Ala.). .. T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: .. Expression of Recombinant VbDFR in E. coli and Purification A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. ..

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Paragraph title: Construction and expression of recombinant human P. carinii MSG. ... For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Paragraph title: Construction of Recombinant cneI Expression Plasmids ... This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Paragraph title: Construction of Recombinant AciI Expression Plasmids ... The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: Subcloning of α-Luffin Gene Fragment After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1
Article Snippet: Recombinant constructs in Escherichia coli [XL1-Blue and BL21(DE3)] were routinely grown and maintained in Luria-Bertani (LB) broth (per liter) containing 10 g of Bacto tryptone, 5 g of yeast extract, and 10 g of NaCl (pH 7.2) or on LB solid medium (1.8% [wt/vol] agar) at 37°C. .. For expression cloning, pET28a (Novagen, Madison, WI) served as the expression vector.

Nucleic Acid Electrophoresis:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: The cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Isolation:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Isolation of recombinant virus, plaque purification, and amplification of high-titer virus stocks were performed according to the manufacturer’s protocols (Invitrogen). .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: .. NUDIX enzyme production Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: Total Acas RNA was isolated using the Qiagen RNeasy Mini Kit and reverse transcribed with SuperScript III First-Strand Synthesis System (Invitrogen, Paisley, UK), using primers AcRegAF and AcRegAR, that contained NheI and EcoRI sites respectively, followed by cDNA amplification with Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA). .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Subcloning:

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: Paragraph title: 3.2. Subcloning of α-Luffin Gene Fragment ... After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. Subcloning of α-Luffin Gene Fragment After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Nickel Column:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site. .. Recombinant protein was solubilized with 6 M urea and purified by affinity chromatography using a nickel column according to the manufacturer’s instructions (Novagen).

Size-exclusion Chromatography:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR cycles consist of an initial denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, annealing at 55 °C for 40 s and extension at 72 °C for 40 sec, and a final extension at 72 °C for 5 min. Sterile deionised water was used as the negative control in all PCR reactions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Sequencing:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site. .. Restriction mapping and sequencing were performed to confirm correct insertion.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes. .. The sequence of both recombinant plasmids was verified by automated Sanger sequencing.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. The sequence of aciI cloned into pDrive and pET28a plasmids was verified by automated Sanger DNA sequencing.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: Plasmid construction A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. To generate the plasmid harboring the codon optimized nucleotide sequence, a codon optimized fragment encoding the first 107 amino acid residues of the N-terminus of the VP1 capsid protein was fused with the C-terminus of the VP1 protein using the 5' and 3' ends of above two DNA fragments and overlapping PCR [ ].

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: 2.1.2 Cloning and expression of Acas RegA The partially assembled Acas genome http://blast.hgsc.bcm.tmc.edu/blast.hgsc?organism=AcastellaniNeff was queried by tBlastn with Ddis RegA, yielding hits on 3 contigs, which after assembly yielded about 3.3 kb of coding sequence homologous to the query sequence, but containing many introns. .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: Subcloning of α-Luffin Gene Fragment After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Affinity Chromatography:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site. .. Recombinant protein was solubilized with 6 M urea and purified by affinity chromatography using a nickel column according to the manufacturer’s instructions (Novagen).

Gel Extraction:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The amplicon with the desired band size was purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pGEMT (Promega, USA), as per the manufacturer’s instructions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Purification:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Recombinant rat P. carinii MSG GP3 (expressed in a baculovirus system) ( ) and purified recombinant β-galactosidase (expressed in the pET28- E. coli system) were used as controls in Western blotting. .. T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The amplicon with the desired band size was purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pGEMT (Promega, USA), as per the manufacturer’s instructions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: .. Expression of Recombinant VbDFR in E. coli and Purification A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. ..

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Isolation of recombinant virus, plaque purification, and amplification of high-titer virus stocks were performed according to the manufacturer’s protocols (Invitrogen). .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes. .. The resulting recombinant plasmid was designated pET28a_cneI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: Paragraph title: Production and purification of ACPs/PCP ... Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

SDS Page:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Paragraph title: SDS-PAGE and Western blotting. ... T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: The cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site.

Plasmid Preparation:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: .. Expression of Recombinant VbDFR in E. coli and Purification A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. ..

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: .. For expression of a conserved region of the MSG in Escherichia coli , the 306-bp PCR product of this region was ligated in frame into pET28A (Novagen, Inc., Madison, Wis.) at the Eco RI site. pET28A is an expression vector in which a histidine tag precedes the insertion site. .. Restriction mapping and sequencing were performed to confirm correct insertion.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The recombinant vector, designated pGEMT-Easy_cneI , was purified and linearized with both NcoI and BamHI enzymes (NEB, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: .. Plasmid construction A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: The resultant products were cloned into pTA2 vector (Toyobo) directly and sequenced to confirm PCR fidelity. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The DNA sequence was determined from three clones and showed an open reading frame of 1863 bp.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: .. Subcloning of α-Luffin Gene Fragment After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1
Article Snippet: .. For expression cloning, pET28a (Novagen, Madison, WI) served as the expression vector. .. Native 2H1NA decarboxylase was purified from crude cell extract of BC1 cells grown for 16 h at 28°C in 4 liters of mineral salt medium (MSM) ( ) containing 0.5 g/liter 2H1NA.

Negative Control:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR cycles consist of an initial denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, annealing at 55 °C for 40 s and extension at 72 °C for 40 sec, and a final extension at 72 °C for 5 min. Sterile deionised water was used as the negative control in all PCR reactions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The PCR cycles used consisted of an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min, and a final extension at 72°C for 5 min. Sterile deionized water was used as the negative control. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Positron Emission Tomography:

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively. .. And the PCP gene was cloned into the pET28a-SUMO (Novagen), resulting in a PCP -containing-plasmid pYY0098.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The DNA sequence was determined from three clones and showed an open reading frame of 1863 bp.

Agarose Gel Electrophoresis:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR product was verified using agarose gel electrophoresis followed by ethidium bromide (Sigma, St. Louis, MO) staining. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Upon running agarose gel electrophoresis, the desired amplicon was extracted using Wizard® SV Gel and PCR Clean-Up System (Promega, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Produced:

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

E. coli Genomic Assay:

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Staining:

Article Title: Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region
Article Snippet: Proteins were stained with Coomassie blue or transferred to nitrocellulose membranes, following which Western blotting was performed with a variety of antisera by standard techniques ( ). .. T7-tag monoclonal antibody, which detects an epitope tag at the amino terminus of the fusion proteins derived from PET28A, was purchased from Novagen.

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR product was verified using agarose gel electrophoresis followed by ethidium bromide (Sigma, St. Louis, MO) staining. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Variant Assay:

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: NUDIX enzyme production Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. Expression constructs of NUDT3 (aa 8–172), NUDT5, the catalytic subunit of NUDT6, NUDT10 (variant AAH50700), NUDT11 (aa 13–164), NUDT12, NUDT14, NUDT15, and NUDT16 (variant AAH31215) in pNIC28 were kind gifts from SGC Stockholm.

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  • 99
    Millipore pet28a
    Pet28a, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pet28a/product/Millipore
    Average 99 stars, based on 718 article reviews
    Price from $9.99 to $1999.99
    pet28a - by Bioz Stars, 2020-01
    99/100 stars
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    85
    Millipore pet28a δn c raf
    RanBPM C-terminal CRA domain directly interacts with <t>ΔN-c-Raf</t> and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p
    Pet28a δn C Raf, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pet28a δn c raf/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pet28a δn c raf - by Bioz Stars, 2020-01
    85/100 stars
      Buy from Supplier

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    RanBPM C-terminal CRA domain directly interacts with ΔN-c-Raf and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: RanBPM C-terminal CRA domain directly interacts with ΔN-c-Raf and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p

    Article Snippet: Plasmid Constructs pCMV-HA-RanBPM and deletion mutants have been previously described [ , ]. pEBG-GST-ΔN-c-Raf was a gift from Dr. Zhijun Luo (Boston University, Boston, MA, USA). pET28a-ΔN-c-Raf was generated by isolating ΔN-c-Raf from pEBG-GST-ΔN-c-Raf and subcloning into pET28a (EMD Millipore, Billerica, MA, USA). pBSK-HA-Ubiquitin was a gift from Dr. Lina Dagnino (Western University, London, ON, Canada). pCGN-HA-RMND5A was obtained by subcloning RMND5A cDNA obtained by RT-PCR from a Jurkat T cell cDNA library into pCGN-HA plasmid.

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Western Blot

    Analysis of RanBPM domains that control C-Raf stability. ( A ) schematic representation of RanBPM mutants. ( B ) and ( C ) Western blot analyses of HeLa RanBPM shRNA cells transfected with pEBG-GST-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs as indicated. c-Raf and HA antibodies were used to detect the levels of ΔN-c-Raf and RanBPM, respectively. β-actin was used as a loading control. A representative Western blot is shown (top) and quantifications of c-Raf levels are shown (bottom graph) with error bars indicating SEM ( n = 5). Deletion of RanBPM C-terminal domain (ΔC1) impairs RanBPM interaction with GST-ΔN-c-Raf. ( D ) and ( E ) GST-Pull-down assays. HeLa RanBPM shRNA cells were transfected with pEBG-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs. ΔN-c-Raf was pulled down through binding to glutathione-sepharose beads and interaction of RanBPM WT and mutants with GST-ΔN-c-Raf assessed by Western blot with an HA antibody. Below: Quantifications were performed by normalizing RanBPM mutant levels to pulled-down GST or GST-ΔN-c-Raf and statistical analyses were performed ( n = 4–7, SEM shown). Different letters are statistically different ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: Analysis of RanBPM domains that control C-Raf stability. ( A ) schematic representation of RanBPM mutants. ( B ) and ( C ) Western blot analyses of HeLa RanBPM shRNA cells transfected with pEBG-GST-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs as indicated. c-Raf and HA antibodies were used to detect the levels of ΔN-c-Raf and RanBPM, respectively. β-actin was used as a loading control. A representative Western blot is shown (top) and quantifications of c-Raf levels are shown (bottom graph) with error bars indicating SEM ( n = 5). Deletion of RanBPM C-terminal domain (ΔC1) impairs RanBPM interaction with GST-ΔN-c-Raf. ( D ) and ( E ) GST-Pull-down assays. HeLa RanBPM shRNA cells were transfected with pEBG-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs. ΔN-c-Raf was pulled down through binding to glutathione-sepharose beads and interaction of RanBPM WT and mutants with GST-ΔN-c-Raf assessed by Western blot with an HA antibody. Below: Quantifications were performed by normalizing RanBPM mutant levels to pulled-down GST or GST-ΔN-c-Raf and statistical analyses were performed ( n = 4–7, SEM shown). Different letters are statistically different ( p

    Article Snippet: Plasmid Constructs pCMV-HA-RanBPM and deletion mutants have been previously described [ , ]. pEBG-GST-ΔN-c-Raf was a gift from Dr. Zhijun Luo (Boston University, Boston, MA, USA). pET28a-ΔN-c-Raf was generated by isolating ΔN-c-Raf from pEBG-GST-ΔN-c-Raf and subcloning into pET28a (EMD Millipore, Billerica, MA, USA). pBSK-HA-Ubiquitin was a gift from Dr. Lina Dagnino (Western University, London, ON, Canada). pCGN-HA-RMND5A was obtained by subcloning RMND5A cDNA obtained by RT-PCR from a Jurkat T cell cDNA library into pCGN-HA plasmid.

    Techniques: Western Blot, shRNA, Transfection, Plasmid Preparation, Mutagenesis, Construct, Binding Assay