Structured Review

Millipore pet28a
<t>GSTe2</t> purification and X-ray three-dimensional structure. (A) Size-exclusion chromatogram of GSTe2 alleles from Benin (BN), Uganda (UG) and Malawi <t>(MAL)</t> alleles. The lines show the absorbance recorded at 280 nm. Molecular-weight markers (Bio-Rad, Hercules, CA, US) are indicated in kilodaltons. (B) SDS-PAGE gel of the three purified GSTe2 alleles (26.8 kDa). (C) Topology of GSTe2 showing the C- and N-terminals, the GSH binding pocket (G-site) and the substrate-binding pocket (H-site). AU, arbitrary units; BN, Benin; GSH, glutathione; MAL, Malawi; MW, molecular weight; UG, Uganda; vol, volume.
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1) Product Images from "A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector"

Article Title: A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

Journal: Genome Biology

doi: 10.1186/gb-2014-15-2-r27

GSTe2 purification and X-ray three-dimensional structure. (A) Size-exclusion chromatogram of GSTe2 alleles from Benin (BN), Uganda (UG) and Malawi (MAL) alleles. The lines show the absorbance recorded at 280 nm. Molecular-weight markers (Bio-Rad, Hercules, CA, US) are indicated in kilodaltons. (B) SDS-PAGE gel of the three purified GSTe2 alleles (26.8 kDa). (C) Topology of GSTe2 showing the C- and N-terminals, the GSH binding pocket (G-site) and the substrate-binding pocket (H-site). AU, arbitrary units; BN, Benin; GSH, glutathione; MAL, Malawi; MW, molecular weight; UG, Uganda; vol, volume.
Figure Legend Snippet: GSTe2 purification and X-ray three-dimensional structure. (A) Size-exclusion chromatogram of GSTe2 alleles from Benin (BN), Uganda (UG) and Malawi (MAL) alleles. The lines show the absorbance recorded at 280 nm. Molecular-weight markers (Bio-Rad, Hercules, CA, US) are indicated in kilodaltons. (B) SDS-PAGE gel of the three purified GSTe2 alleles (26.8 kDa). (C) Topology of GSTe2 showing the C- and N-terminals, the GSH binding pocket (G-site) and the substrate-binding pocket (H-site). AU, arbitrary units; BN, Benin; GSH, glutathione; MAL, Malawi; MW, molecular weight; UG, Uganda; vol, volume.

Techniques Used: Purification, Molecular Weight, SDS Page, Binding Assay

GSTe2 polymorphism and DDT resistance. (A) Maximum likelihood tree of GSTe2 cDNA across Africa. (B) Same as in (A), but with DDT-resistant (AL) and DDT-susceptible (DE) mosquitoes in Benin (genomic DNA). (C) GSTe2 haplotype network (TCS) between susceptible and resistant mosquitoes (Benin). The polygon size reflects the haplotype frequency. Each node represents a mutation (number). (D) GSTe2 expression profile of the three L119F genotypes in Cameroon (Gounougou). BN, Benin; DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; UG, Uganda; ZB; Zambia.
Figure Legend Snippet: GSTe2 polymorphism and DDT resistance. (A) Maximum likelihood tree of GSTe2 cDNA across Africa. (B) Same as in (A), but with DDT-resistant (AL) and DDT-susceptible (DE) mosquitoes in Benin (genomic DNA). (C) GSTe2 haplotype network (TCS) between susceptible and resistant mosquitoes (Benin). The polygon size reflects the haplotype frequency. Each node represents a mutation (number). (D) GSTe2 expression profile of the three L119F genotypes in Cameroon (Gounougou). BN, Benin; DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; UG, Uganda; ZB; Zambia.

Techniques Used: Mutagenesis, Expressing

Maximum likelihood phylogenetic tree of GSTe2 (coding and non-coding regions) across Africa. The analysis involved 92 sequences (2n) labeled. All positions with gaps and missing data were eliminated. There were a total of 881 positions in the final dataset. A resistant clade that is less polymorphic and dominated by the Benin population is clearly identifiable to the right of the tree whereas a susceptible clade that is more polymorphic and more cosmopolitan is at the left of the tree. BN, Benin; CAM, Cameroon; GH, Ghana; MAL, Malawi; MOZ, Mozambique; UG, Uganda.
Figure Legend Snippet: Maximum likelihood phylogenetic tree of GSTe2 (coding and non-coding regions) across Africa. The analysis involved 92 sequences (2n) labeled. All positions with gaps and missing data were eliminated. There were a total of 881 positions in the final dataset. A resistant clade that is less polymorphic and dominated by the Benin population is clearly identifiable to the right of the tree whereas a susceptible clade that is more polymorphic and more cosmopolitan is at the left of the tree. BN, Benin; CAM, Cameroon; GH, Ghana; MAL, Malawi; MOZ, Mozambique; UG, Uganda.

Techniques Used: Labeling, Chick Chorioallantoic Membrane Assay

GSTe2 expression and functional analysis. (A) Comparative qRT-PCR examining DDT-resistant (Benin) and DDT-susceptible (Mozambique, Malawi and Uganda) mosquitoes. (B) DDT bioassay tests on transgenic Act5C-GSTe2 flies (Exp-GSTe2) and control strains (two parental (UAS-GSTe2 and GAL4-actin) and F 1 progeny that do not express the GSTe2 transgene (Cont-NO)). (C) The same bioassays with permethrin. (D) DDT metabolic activity (depletion rate after 1 hr) of GSTe2 alleles (mean ± standard deviation). (E) Michaelis–Menten enzyme kinetics for resistant and susceptible GSTe2 alleles (F) Permethrin metabolic activity (depletion rate after 1 hr) for the 119 F GSTe2 allele (mean ± standard deviation). DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; MOZ, Mozambique; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; UG, Uganda, DDE, dichlorodiphenyldichloroethylene.
Figure Legend Snippet: GSTe2 expression and functional analysis. (A) Comparative qRT-PCR examining DDT-resistant (Benin) and DDT-susceptible (Mozambique, Malawi and Uganda) mosquitoes. (B) DDT bioassay tests on transgenic Act5C-GSTe2 flies (Exp-GSTe2) and control strains (two parental (UAS-GSTe2 and GAL4-actin) and F 1 progeny that do not express the GSTe2 transgene (Cont-NO)). (C) The same bioassays with permethrin. (D) DDT metabolic activity (depletion rate after 1 hr) of GSTe2 alleles (mean ± standard deviation). (E) Michaelis–Menten enzyme kinetics for resistant and susceptible GSTe2 alleles (F) Permethrin metabolic activity (depletion rate after 1 hr) for the 119 F GSTe2 allele (mean ± standard deviation). DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; MOZ, Mozambique; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; UG, Uganda, DDE, dichlorodiphenyldichloroethylene.

Techniques Used: Expressing, Functional Assay, Quantitative RT-PCR, Transgenic Assay, Activity Assay, Standard Deviation, Polymerase Chain Reaction

2) 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

3) 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

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 "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

6) 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

7) 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

8) 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

9) Product Images from "Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli"

Article Title: Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli

Journal: Microbial Cell Factories

doi: 10.1186/s12934-015-0230-8

Cell lysis determination and inactive Cel-CD secretion analysis. (A) Immunoblotting detection of Cel-CD, GroEL and MBP in the culture medium. Samples were subjected to immunoblotting with an anti-Cel-CD antibody, an anti-GroEL antibody or an anti-MBP antibody. (B) SDS analysis of cellulase (wt) and its mutants (E160Q, E160Q E274Q) secreted into the culture medium. The activities of the secreted mutants of Cel-CD (E160Q, E160Q E274Q) were analyzed by Congo red staining method (4 h, 6 h, 8 h, 16 h). (C) TEM images of E. coli BL21 (DE3) that containing different plasmids. (1) pET28a/cel, (2) pET28a/E160Q, (3) pET28a/E160Q E274Q, (4) pET28a. Protein samples were separated on 12% SDS-PAGE. Aliquots corresponding to 20 μL of the culture medium were loaded onto the gel. Molecular size markers are shown in kDa. Lane M is the marker; Lane C is the control of collected cells.
Figure Legend Snippet: Cell lysis determination and inactive Cel-CD secretion analysis. (A) Immunoblotting detection of Cel-CD, GroEL and MBP in the culture medium. Samples were subjected to immunoblotting with an anti-Cel-CD antibody, an anti-GroEL antibody or an anti-MBP antibody. (B) SDS analysis of cellulase (wt) and its mutants (E160Q, E160Q E274Q) secreted into the culture medium. The activities of the secreted mutants of Cel-CD (E160Q, E160Q E274Q) were analyzed by Congo red staining method (4 h, 6 h, 8 h, 16 h). (C) TEM images of E. coli BL21 (DE3) that containing different plasmids. (1) pET28a/cel, (2) pET28a/E160Q, (3) pET28a/E160Q E274Q, (4) pET28a. Protein samples were separated on 12% SDS-PAGE. Aliquots corresponding to 20 μL of the culture medium were loaded onto the gel. Molecular size markers are shown in kDa. Lane M is the marker; Lane C is the control of collected cells.

Techniques Used: Lysis, Staining, Transmission Electron Microscopy, SDS Page, Marker

10) 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

11) 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

12) 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

13) 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

14) 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

15) 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

16) 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

17) Product Images from "Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase"

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase

Journal: Avicenna Journal of Medical Biotechnology

doi:

SDS-PAGE analysis of recombinant creatinase expression. Lane 1 and 2 are soluble fractions of induced BL21 (containing pET28a-cre) for 4 and 16 hr and 3 is soluble fraction of induced negative control. Lane 4 is the protein ladder. Lane 5 and 6 are crude extract of total protein of induced BL21 (containing pET28a-cre), and lane 7 is the negative control.
Figure Legend Snippet: SDS-PAGE analysis of recombinant creatinase expression. Lane 1 and 2 are soluble fractions of induced BL21 (containing pET28a-cre) for 4 and 16 hr and 3 is soluble fraction of induced negative control. Lane 4 is the protein ladder. Lane 5 and 6 are crude extract of total protein of induced BL21 (containing pET28a-cre), and lane 7 is the negative control.

Techniques Used: SDS Page, Recombinant, Expressing, Negative Control

Lane 1: PCR product of Cre gene, lane 2: pET28a-cre plasmid extraction result, lane 3: DNA ladder, lane 4: double digestion of recombinant pET28a-cre by NheI and XhoI. Products were electrophoresed on 0.7% agarose gel.
Figure Legend Snippet: Lane 1: PCR product of Cre gene, lane 2: pET28a-cre plasmid extraction result, lane 3: DNA ladder, lane 4: double digestion of recombinant pET28a-cre by NheI and XhoI. Products were electrophoresed on 0.7% agarose gel.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Agarose Gel Electrophoresis

18) Product Images from "Specific DNA Binding and Regulation of Its Own Expression by the AidB Protein in Escherichia coli ▿"

Article Title: Specific DNA Binding and Regulation of Its Own Expression by the AidB Protein in Escherichia coli ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00858-10

(A) Gel retardation experiments carried out by incubating the AidB, AidB I-III, and AidB IV proteins with the UP35 P aidB probe. Lane 1, UP35 P aidB probe; lanes 2 to 4, UP35 P aidB fragment incubated with the AidB I-III, AidB, and AidB IV proteins, respectively. The two shifted complexes obtained using AidB IV could be due to multiple monomers binding the UP35 P aidB probe. (B) In vivo transcription from the aidB promoter. The vector pMV132H was introduced into the E. coli strain MV1161 (wild type) or MV5924 (Δ aidB ). Strain MV5924, carrying pMV132H, was also individually transformed with pET28a-P lac - aidB , pET28a-P lac - aidB ′I-III', or pET28a-P lac - aidB ′IV', and the corresponding β-galactosidase activity was evaluated in the exponential phase. Means and standard deviations have been calculated with results from four independent assays.
Figure Legend Snippet: (A) Gel retardation experiments carried out by incubating the AidB, AidB I-III, and AidB IV proteins with the UP35 P aidB probe. Lane 1, UP35 P aidB probe; lanes 2 to 4, UP35 P aidB fragment incubated with the AidB I-III, AidB, and AidB IV proteins, respectively. The two shifted complexes obtained using AidB IV could be due to multiple monomers binding the UP35 P aidB probe. (B) In vivo transcription from the aidB promoter. The vector pMV132H was introduced into the E. coli strain MV1161 (wild type) or MV5924 (Δ aidB ). Strain MV5924, carrying pMV132H, was also individually transformed with pET28a-P lac - aidB , pET28a-P lac - aidB ′I-III', or pET28a-P lac - aidB ′IV', and the corresponding β-galactosidase activity was evaluated in the exponential phase. Means and standard deviations have been calculated with results from four independent assays.

Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, In Vivo, Plasmid Preparation, Transformation Assay, Activity Assay

In vivo transcription from the aidB promoter. The vector pMV132H was introduced into the E. coli strains MV1161 (wild type) and MV5924 (Δ aidB ),and the β-galactosidase specific activity was monitored in the exponential phase. Strain MV5924 was also transformed with the pET28a-P lac - aidB plasmid, producing a functional AidB protein, and the corresponding β-galactosidase activity was evaluated. Means and standard deviations have been calculated from four independent assays.
Figure Legend Snippet: In vivo transcription from the aidB promoter. The vector pMV132H was introduced into the E. coli strains MV1161 (wild type) and MV5924 (Δ aidB ),and the β-galactosidase specific activity was monitored in the exponential phase. Strain MV5924 was also transformed with the pET28a-P lac - aidB plasmid, producing a functional AidB protein, and the corresponding β-galactosidase activity was evaluated. Means and standard deviations have been calculated from four independent assays.

Techniques Used: In Vivo, Plasmid Preparation, Activity Assay, Transformation Assay, Functional Assay

19) 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

20) 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

21) Product Images from "Construction and Identification of a Recombinant Plasmid Encoding Echinococcus granulosus Oncosphere Antigen (EG95)"

Article Title: Construction and Identification of a Recombinant Plasmid Encoding Echinococcus granulosus Oncosphere Antigen (EG95)

Journal: Iranian Journal of Parasitology

doi:

Identification of EG95 expression in E.coli BL21 (DE3) pLysS bacteria by (A) SDS-PAGE and (B) western-blotting. M: prestained protein marker. A: lane 1: Non-induced bacteria transfected with pET28a- EG95 ; 2: 1-hour induced bacteria transfected with pET28a- EG95 with 1 mmol/L IPTG; 3: 2-hour induced bacteria transfected with pET28a- EG95 ; 4: 3-hour induced bacteria transfected with pET28a- EG95 ; 5: 4-hour induced bacteria transfected with pET28a- EG95 ; 6: 5-hour induced bacteria transfected with pET28a- EG95; 7: Bacteria transfected with empty pET28. B: Lane 1: rEG95-His/pET28a detected with the anti-His monoclonal antibodies
Figure Legend Snippet: Identification of EG95 expression in E.coli BL21 (DE3) pLysS bacteria by (A) SDS-PAGE and (B) western-blotting. M: prestained protein marker. A: lane 1: Non-induced bacteria transfected with pET28a- EG95 ; 2: 1-hour induced bacteria transfected with pET28a- EG95 with 1 mmol/L IPTG; 3: 2-hour induced bacteria transfected with pET28a- EG95 ; 4: 3-hour induced bacteria transfected with pET28a- EG95 ; 5: 4-hour induced bacteria transfected with pET28a- EG95 ; 6: 5-hour induced bacteria transfected with pET28a- EG95; 7: Bacteria transfected with empty pET28. B: Lane 1: rEG95-His/pET28a detected with the anti-His monoclonal antibodies

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

22) 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

23) Product Images from "Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria"

Article Title: Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA117.000246

Epoxide hydrolase activity of EphA–EphG proteins in vitro . A , reaction catalyzed by EH. B , analysis of recombinant protein production in E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephD tb , BL21(DE3) pET29a- ephD tb -EH , BL21(DE3) pET29a- ephD tb -SDR , BL21(DE3) pET29a- ephD smeg , BL21(DE3) pET29a- ephA tb , BL21(AI) pDEST17- ephB tb , BL21(DE3) pET29a- ephC tb , BL21(AI) pET28a- ephE tb , and BL21(DE3) pET28a- ephF tb . (The line with recombinant EphG is not shown because the signal was hardly visible.) SDS-PAGE analysis of 10,000 × g supernatants of cell lysates, 60 μg of proteins were loaded per lane. Recombinant proteins were detected by Western blotting analysis and immunodetection with anti-His antibodies. C , TLC analysis of the reaction products resulting from the incubation of [ 14 C]9,10- cis -epoxystearic acid with E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephD tb , or BL21(DE3) pET29a- ephD smeg fractions for 5–120 min at 37 °C and E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephD tb -EH , BL21(DE3) pET29a- ephD tb -SDR , and BL21(DE3) pET29a- ephD tb for 60 min. The plates were developed using n -hexane:diethyl ether:formic acid (70:30:2) and exposed to autoradiography film at −80 °C for 2–5 days. D , TLC analysis of the products of the EH assays containing 10,000 × g supernatants of lysates of E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephA tb , BL21(AI) pDEST17- ephB tb , BL21 (AI) pET28a- ephE tb , BL21(DE3) pET28a- ephF tb , and BL21(DE3) pET29a- ephG tb as the enzymatic sources. The reactions ran for 1 h at 37 °C. The plates were developed and exposed as described above.
Figure Legend Snippet: Epoxide hydrolase activity of EphA–EphG proteins in vitro . A , reaction catalyzed by EH. B , analysis of recombinant protein production in E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephD tb , BL21(DE3) pET29a- ephD tb -EH , BL21(DE3) pET29a- ephD tb -SDR , BL21(DE3) pET29a- ephD smeg , BL21(DE3) pET29a- ephA tb , BL21(AI) pDEST17- ephB tb , BL21(DE3) pET29a- ephC tb , BL21(AI) pET28a- ephE tb , and BL21(DE3) pET28a- ephF tb . (The line with recombinant EphG is not shown because the signal was hardly visible.) SDS-PAGE analysis of 10,000 × g supernatants of cell lysates, 60 μg of proteins were loaded per lane. Recombinant proteins were detected by Western blotting analysis and immunodetection with anti-His antibodies. C , TLC analysis of the reaction products resulting from the incubation of [ 14 C]9,10- cis -epoxystearic acid with E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephD tb , or BL21(DE3) pET29a- ephD smeg fractions for 5–120 min at 37 °C and E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephD tb -EH , BL21(DE3) pET29a- ephD tb -SDR , and BL21(DE3) pET29a- ephD tb for 60 min. The plates were developed using n -hexane:diethyl ether:formic acid (70:30:2) and exposed to autoradiography film at −80 °C for 2–5 days. D , TLC analysis of the products of the EH assays containing 10,000 × g supernatants of lysates of E. coli BL21(DE3) pET29a, BL21(DE3) pET29a- ephA tb , BL21(AI) pDEST17- ephB tb , BL21 (AI) pET28a- ephE tb , BL21(DE3) pET28a- ephF tb , and BL21(DE3) pET29a- ephG tb as the enzymatic sources. The reactions ran for 1 h at 37 °C. The plates were developed and exposed as described above.

Techniques Used: Activity Assay, In Vitro, Recombinant, SDS Page, Western Blot, Immunodetection, Thin Layer Chromatography, Incubation, Autoradiography

24) 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

25) 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

26) Product Images from "A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector"

Article Title: A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

Journal: Genome Biology

doi: 10.1186/gb-2014-15-2-r27

GSTe2 purification and X-ray three-dimensional structure. (A) Size-exclusion chromatogram of GSTe2 alleles from Benin (BN), Uganda (UG) and Malawi (MAL) alleles. The lines show the absorbance recorded at 280 nm. Molecular-weight markers (Bio-Rad, Hercules, CA, US) are indicated in kilodaltons. (B) SDS-PAGE gel of the three purified GSTe2 alleles (26.8 kDa). (C) Topology of GSTe2 showing the C- and N-terminals, the GSH binding pocket (G-site) and the substrate-binding pocket (H-site). AU, arbitrary units; BN, Benin; GSH, glutathione; MAL, Malawi; MW, molecular weight; UG, Uganda; vol, volume.
Figure Legend Snippet: GSTe2 purification and X-ray three-dimensional structure. (A) Size-exclusion chromatogram of GSTe2 alleles from Benin (BN), Uganda (UG) and Malawi (MAL) alleles. The lines show the absorbance recorded at 280 nm. Molecular-weight markers (Bio-Rad, Hercules, CA, US) are indicated in kilodaltons. (B) SDS-PAGE gel of the three purified GSTe2 alleles (26.8 kDa). (C) Topology of GSTe2 showing the C- and N-terminals, the GSH binding pocket (G-site) and the substrate-binding pocket (H-site). AU, arbitrary units; BN, Benin; GSH, glutathione; MAL, Malawi; MW, molecular weight; UG, Uganda; vol, volume.

Techniques Used: Purification, Molecular Weight, SDS Page, Binding Assay

GSTe2 polymorphism and DDT resistance. (A) Maximum likelihood tree of GSTe2 cDNA across Africa. (B) Same as in (A), but with DDT-resistant (AL) and DDT-susceptible (DE) mosquitoes in Benin (genomic DNA). (C) GSTe2 haplotype network (TCS) between susceptible and resistant mosquitoes (Benin). The polygon size reflects the haplotype frequency. Each node represents a mutation (number). (D) GSTe2 expression profile of the three L119F genotypes in Cameroon (Gounougou). BN, Benin; DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; UG, Uganda; ZB; Zambia.
Figure Legend Snippet: GSTe2 polymorphism and DDT resistance. (A) Maximum likelihood tree of GSTe2 cDNA across Africa. (B) Same as in (A), but with DDT-resistant (AL) and DDT-susceptible (DE) mosquitoes in Benin (genomic DNA). (C) GSTe2 haplotype network (TCS) between susceptible and resistant mosquitoes (Benin). The polygon size reflects the haplotype frequency. Each node represents a mutation (number). (D) GSTe2 expression profile of the three L119F genotypes in Cameroon (Gounougou). BN, Benin; DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; UG, Uganda; ZB; Zambia.

Techniques Used: Mutagenesis, Expressing

Maximum likelihood phylogenetic tree of GSTe2 (coding and non-coding regions) across Africa. The analysis involved 92 sequences (2n) labeled. All positions with gaps and missing data were eliminated. There were a total of 881 positions in the final dataset. A resistant clade that is less polymorphic and dominated by the Benin population is clearly identifiable to the right of the tree whereas a susceptible clade that is more polymorphic and more cosmopolitan is at the left of the tree. BN, Benin; CAM, Cameroon; GH, Ghana; MAL, Malawi; MOZ, Mozambique; UG, Uganda.
Figure Legend Snippet: Maximum likelihood phylogenetic tree of GSTe2 (coding and non-coding regions) across Africa. The analysis involved 92 sequences (2n) labeled. All positions with gaps and missing data were eliminated. There were a total of 881 positions in the final dataset. A resistant clade that is less polymorphic and dominated by the Benin population is clearly identifiable to the right of the tree whereas a susceptible clade that is more polymorphic and more cosmopolitan is at the left of the tree. BN, Benin; CAM, Cameroon; GH, Ghana; MAL, Malawi; MOZ, Mozambique; UG, Uganda.

Techniques Used: Labeling, Chick Chorioallantoic Membrane Assay

GSTe2 expression and functional analysis. (A) Comparative qRT-PCR examining DDT-resistant (Benin) and DDT-susceptible (Mozambique, Malawi and Uganda) mosquitoes. (B) DDT bioassay tests on transgenic Act5C-GSTe2 flies (Exp-GSTe2) and control strains (two parental (UAS-GSTe2 and GAL4-actin) and F 1 progeny that do not express the GSTe2 transgene (Cont-NO)). (C) The same bioassays with permethrin. (D) DDT metabolic activity (depletion rate after 1 hr) of GSTe2 alleles (mean ± standard deviation). (E) Michaelis–Menten enzyme kinetics for resistant and susceptible GSTe2 alleles (F) Permethrin metabolic activity (depletion rate after 1 hr) for the 119 F GSTe2 allele (mean ± standard deviation). DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; MOZ, Mozambique; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; UG, Uganda, DDE, dichlorodiphenyldichloroethylene.
Figure Legend Snippet: GSTe2 expression and functional analysis. (A) Comparative qRT-PCR examining DDT-resistant (Benin) and DDT-susceptible (Mozambique, Malawi and Uganda) mosquitoes. (B) DDT bioassay tests on transgenic Act5C-GSTe2 flies (Exp-GSTe2) and control strains (two parental (UAS-GSTe2 and GAL4-actin) and F 1 progeny that do not express the GSTe2 transgene (Cont-NO)). (C) The same bioassays with permethrin. (D) DDT metabolic activity (depletion rate after 1 hr) of GSTe2 alleles (mean ± standard deviation). (E) Michaelis–Menten enzyme kinetics for resistant and susceptible GSTe2 alleles (F) Permethrin metabolic activity (depletion rate after 1 hr) for the 119 F GSTe2 allele (mean ± standard deviation). DDT, dichlorodiphenyltrichloroethane; MAL, Malawi; MOZ, Mozambique; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; UG, Uganda, DDE, dichlorodiphenyldichloroethylene.

Techniques Used: Expressing, Functional Assay, Quantitative RT-PCR, Transgenic Assay, Activity Assay, Standard Deviation, Polymerase Chain Reaction

27) Product Images from "Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system"

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system

Journal: Iranian Journal of Veterinary Research

doi:

Cloning of HA1 cDNA into pET28a. (A) Schematic illustration of pET28a-HA1 construct contained optimized HA1 antigen. (B) Construct map of recombinant pET28a-HA1 vector
Figure Legend Snippet: Cloning of HA1 cDNA into pET28a. (A) Schematic illustration of pET28a-HA1 construct contained optimized HA1 antigen. (B) Construct map of recombinant pET28a-HA1 vector

Techniques Used: Clone Assay, Construct, Recombinant, Plasmid Preparation

Construction and cloning of HA1 protein. (A) Digestion of pUC57 vector containing HA1 gene by enzymes BamHI and XhoI. M: Molecular marker. Lanes 1 and 2: Digested plasmid and HA1 gene. (B) Digestion of pET28a-HA1 vector by enzymes BamHI and XhoI. M: Molecular marker. Lane 1: Digested pET and HA1 gene. (C) PCR analysis for detection of HA1 gene in transformed E. coli clonies contained pET28a-HA1 construct by HA1 specific primers. M: Molecular marker. Lane 1: Positive control, Lane 2: Transformed bacterial clony (The 329 bp band was clear), and Lane 3: Negative control
Figure Legend Snippet: Construction and cloning of HA1 protein. (A) Digestion of pUC57 vector containing HA1 gene by enzymes BamHI and XhoI. M: Molecular marker. Lanes 1 and 2: Digested plasmid and HA1 gene. (B) Digestion of pET28a-HA1 vector by enzymes BamHI and XhoI. M: Molecular marker. Lane 1: Digested pET and HA1 gene. (C) PCR analysis for detection of HA1 gene in transformed E. coli clonies contained pET28a-HA1 construct by HA1 specific primers. M: Molecular marker. Lane 1: Positive control, Lane 2: Transformed bacterial clony (The 329 bp band was clear), and Lane 3: Negative control

Techniques Used: Clone Assay, Plasmid Preparation, Marker, Positron Emission Tomography, Polymerase Chain Reaction, Transformation Assay, Construct, Positive Control, Negative Control

Optimizing the expression of HA1 protein. (A) Time-course induction study in Escherichia coli strain BL21 (DE3). Expression of HA1 protein was induced by 1 mM IPTG. Lane M: Protein ladder. Lanes 1-7: Harvested cell aggregate ( E. coli BL21 pet28a-HA1) at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h, respectively; Lanes 8 and 9: Negative control samples ( E. coli BL21 pet28a+) at 0 h and 6 h, respectively. (B) The best IPTG concentration study after 6 h induction. Lane M: Protein ladder. Lanes 1-5: Control samples ( E. coli BL21 pet28a+) with 0, 0.2, 0.4, 0.8 and 1 Mm IPTG concentration, respectively. Lane 6-10: HA1 expressed samples ( E. coli BL21 pet28a-HA1) with 0, 0.2, 0.8, 1 and 0.4 mM IPTG concentration, respectively
Figure Legend Snippet: Optimizing the expression of HA1 protein. (A) Time-course induction study in Escherichia coli strain BL21 (DE3). Expression of HA1 protein was induced by 1 mM IPTG. Lane M: Protein ladder. Lanes 1-7: Harvested cell aggregate ( E. coli BL21 pet28a-HA1) at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h, respectively; Lanes 8 and 9: Negative control samples ( E. coli BL21 pet28a+) at 0 h and 6 h, respectively. (B) The best IPTG concentration study after 6 h induction. Lane M: Protein ladder. Lanes 1-5: Control samples ( E. coli BL21 pet28a+) with 0, 0.2, 0.4, 0.8 and 1 Mm IPTG concentration, respectively. Lane 6-10: HA1 expressed samples ( E. coli BL21 pet28a-HA1) with 0, 0.2, 0.8, 1 and 0.4 mM IPTG concentration, respectively

Techniques Used: Expressing, Negative Control, Concentration Assay

28) 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

29) 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

30) 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

31) Product Images from "A Blood Fluke Serine Protease Inhibitor Regulates an Endogenous Larval Elastase *"

Article Title: A Blood Fluke Serine Protease Inhibitor Regulates an Endogenous Larval Elastase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.313304

35 S-SmSrpQ complex. a , SmSrpQ was cloned into pET28a+ and expressed using the rabbit reticulolysate system in the presence of [ 35 S]methionine yielding a protein of ∼47 kDa. When incubated with cercarial lysate (at 25 °C, 30 °C,
Figure Legend Snippet: 35 S-SmSrpQ complex. a , SmSrpQ was cloned into pET28a+ and expressed using the rabbit reticulolysate system in the presence of [ 35 S]methionine yielding a protein of ∼47 kDa. When incubated with cercarial lysate (at 25 °C, 30 °C,

Techniques Used: Clone Assay, Incubation

32) 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

33) Product Images from "Cloning, Expression, and Structural Elucidation of a Biotechnologically Potential Alkaline Serine Protease From a Newly Isolated Haloalkaliphilic Bacillus lehensis JO-26"

Article Title: Cloning, Expression, and Structural Elucidation of a Biotechnologically Potential Alkaline Serine Protease From a Newly Isolated Haloalkaliphilic Bacillus lehensis JO-26

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.00941

(A) Protein expression profile of APrBL. M: 15–150 KD, Lane 1: Preinduced soluble fraction of Escherichia coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 2: Preinduced insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 3: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, Lane 4: Insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, Lane 5: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 1 mM IPTG, Lane 6: Insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 1 mM IPTG, Lane 7: Soluble fraction of E. coli BL21 (DE3), Lane 8: Insoluble fraction of E. coli BL21 (DE3), Lane 9: Uninduced soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 10: Uninduced insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a. (B) Protein purification using Ni-NTA column after expression of the protein in pET28a, 28°C, 0.2 mM IPTG. M: 15–150 KD, L: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, FT: Flow through from the column, E: Column elution in 250 mM imidazole.
Figure Legend Snippet: (A) Protein expression profile of APrBL. M: 15–150 KD, Lane 1: Preinduced soluble fraction of Escherichia coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 2: Preinduced insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 3: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, Lane 4: Insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, Lane 5: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 1 mM IPTG, Lane 6: Insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 1 mM IPTG, Lane 7: Soluble fraction of E. coli BL21 (DE3), Lane 8: Insoluble fraction of E. coli BL21 (DE3), Lane 9: Uninduced soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a, Lane 10: Uninduced insoluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a. (B) Protein purification using Ni-NTA column after expression of the protein in pET28a, 28°C, 0.2 mM IPTG. M: 15–150 KD, L: Soluble fraction of E. coli BL21 (DE3) harboring recombinant APrBL-pET28a induced with 0.2 mM IPTG, FT: Flow through from the column, E: Column elution in 250 mM imidazole.

Techniques Used: Expressing, Recombinant, Protein Purification

34) 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

35) 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

36) 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

37) 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

38) 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

39) 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

40) 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

Related Articles

Clone Assay:

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Positron Emission Tomography:

Article Title: Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Article Snippet: .. Recombinant protein purification FLAG-EGFP and FLAG-EGFP-Tsg101-CTD (both with the A206K mutation in EGFP that renders it monomeric), as well as full length NiV-C (native sequence) with a N-terminal HA tag, were inserted into the pET-15b vector (Novagen), which appends an additional N-terminal 6XHis tag. .. The recombinant proteins were purified from BL21(DE3) E . coli as previously described [ ] with minor modifications.

In Vitro:

Article Title: Cytoplasmic Localization of p21 Protects Trophoblast Giant Cells from DNA Damage Induced Apoptosis
Article Snippet: .. Protein Kinase Assays Recombinant mouse p21 proteins were purified from bacteria and tested as Akt1 (Sigma SRP-0353-10UG) substrates using an in vitro kinase assay previously described . .. shRNA Suppression of Akt1 and p27 Akt1 and p27 shRNA oligonucleotides were designed against three different sequences within their respective target RNA ( ) and cloned into pLKO.1-TRC (Addgene plasmid 10878, ).

Expressing:

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Mutagenesis:

Article Title: Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein
Article Snippet: .. Recombinant protein purification Flag-tagged XPA and HECT domain of HERC2 (wild-type and Cys→Ala mutant) were immunopurified using anti-Flag agarose (Sigma). ..

Article Title: Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Article Snippet: .. Recombinant protein purification FLAG-EGFP and FLAG-EGFP-Tsg101-CTD (both with the A206K mutation in EGFP that renders it monomeric), as well as full length NiV-C (native sequence) with a N-terminal HA tag, were inserted into the pET-15b vector (Novagen), which appends an additional N-terminal 6XHis tag. .. The recombinant proteins were purified from BL21(DE3) E . coli as previously described [ ] with minor modifications.

Incubation:

Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties
Article Snippet: .. Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent. .. The samples were submitted to electrophoresis on an 8%–15% SDS-PAGE gel and stained with Coomassie blue.

Article Title: Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock *
Article Snippet: .. Pulldown Assay for Protein-Protein Interactions Purified recombinant proteins at 0.1 nm were mixed in 200 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 100 μg/ml of BSA, 0.05% Nonidet P-40) and incubated at 22 °C for 15 min. Then 20 μl of either FLAG- or V5-agarose (Sigma) beads were added and the mixture was incubated at 4 °C for 90 min. Then, the beads were washed with 1 ml of ice-cold binding buffer 4 times and after the final wash resuspended in 25 μl of SDS loading buffer and heated at 95 °C for 5 min. .. The proteins were then separated on SDS-PAGE and visualized by immunoblotting.

Infection:

Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
Article Snippet: .. Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

Purification:

Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties
Article Snippet: .. Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent. .. The samples were submitted to electrophoresis on an 8%–15% SDS-PAGE gel and stained with Coomassie blue.

Article Title: Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock *
Article Snippet: .. Pulldown Assay for Protein-Protein Interactions Purified recombinant proteins at 0.1 nm were mixed in 200 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 100 μg/ml of BSA, 0.05% Nonidet P-40) and incubated at 22 °C for 15 min. Then 20 μl of either FLAG- or V5-agarose (Sigma) beads were added and the mixture was incubated at 4 °C for 90 min. Then, the beads were washed with 1 ml of ice-cold binding buffer 4 times and after the final wash resuspended in 25 μl of SDS loading buffer and heated at 95 °C for 5 min. .. The proteins were then separated on SDS-PAGE and visualized by immunoblotting.

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Article Title: Cytoplasmic Localization of p21 Protects Trophoblast Giant Cells from DNA Damage Induced Apoptosis
Article Snippet: .. Protein Kinase Assays Recombinant mouse p21 proteins were purified from bacteria and tested as Akt1 (Sigma SRP-0353-10UG) substrates using an in vitro kinase assay previously described . .. shRNA Suppression of Akt1 and p27 Akt1 and p27 shRNA oligonucleotides were designed against three different sequences within their respective target RNA ( ) and cloned into pLKO.1-TRC (Addgene plasmid 10878, ).

Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
Article Snippet: .. Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Mass spectrometry analysis was performed on Coomassie-stained gel bands corresponding to the readthrough GST proteins.

Protein Purification:

Article Title: Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein
Article Snippet: .. Recombinant protein purification Flag-tagged XPA and HECT domain of HERC2 (wild-type and Cys→Ala mutant) were immunopurified using anti-Flag agarose (Sigma). ..

Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
Article Snippet: .. Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

Article Title: Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Article Snippet: .. Recombinant protein purification FLAG-EGFP and FLAG-EGFP-Tsg101-CTD (both with the A206K mutation in EGFP that renders it monomeric), as well as full length NiV-C (native sequence) with a N-terminal HA tag, were inserted into the pET-15b vector (Novagen), which appends an additional N-terminal 6XHis tag. .. The recombinant proteins were purified from BL21(DE3) E . coli as previously described [ ] with minor modifications.

Nuclear Magnetic Resonance:

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Labeling:

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Mass Spectrometry:

Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
Article Snippet: .. Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Mass spectrometry analysis was performed on Coomassie-stained gel bands corresponding to the readthrough GST proteins.

Sequencing:

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Article Title: Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Article Snippet: .. Recombinant protein purification FLAG-EGFP and FLAG-EGFP-Tsg101-CTD (both with the A206K mutation in EGFP that renders it monomeric), as well as full length NiV-C (native sequence) with a N-terminal HA tag, were inserted into the pET-15b vector (Novagen), which appends an additional N-terminal 6XHis tag. .. The recombinant proteins were purified from BL21(DE3) E . coli as previously described [ ] with minor modifications.

Recombinant:

Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties
Article Snippet: .. Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent. .. The samples were submitted to electrophoresis on an 8%–15% SDS-PAGE gel and stained with Coomassie blue.

Article Title: Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock *
Article Snippet: .. Pulldown Assay for Protein-Protein Interactions Purified recombinant proteins at 0.1 nm were mixed in 200 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 100 μg/ml of BSA, 0.05% Nonidet P-40) and incubated at 22 °C for 15 min. Then 20 μl of either FLAG- or V5-agarose (Sigma) beads were added and the mixture was incubated at 4 °C for 90 min. Then, the beads were washed with 1 ml of ice-cold binding buffer 4 times and after the final wash resuspended in 25 μl of SDS loading buffer and heated at 95 °C for 5 min. .. The proteins were then separated on SDS-PAGE and visualized by immunoblotting.

Article Title: Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein
Article Snippet: .. Recombinant protein purification Flag-tagged XPA and HECT domain of HERC2 (wild-type and Cys→Ala mutant) were immunopurified using anti-Flag agarose (Sigma). ..

Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
Article Snippet: .. Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

Article Title: Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Article Snippet: .. Recombinant protein purification FLAG-EGFP and FLAG-EGFP-Tsg101-CTD (both with the A206K mutation in EGFP that renders it monomeric), as well as full length NiV-C (native sequence) with a N-terminal HA tag, were inserted into the pET-15b vector (Novagen), which appends an additional N-terminal 6XHis tag. .. The recombinant proteins were purified from BL21(DE3) E . coli as previously described [ ] with minor modifications.

Article Title: Cytoplasmic Localization of p21 Protects Trophoblast Giant Cells from DNA Damage Induced Apoptosis
Article Snippet: .. Protein Kinase Assays Recombinant mouse p21 proteins were purified from bacteria and tested as Akt1 (Sigma SRP-0353-10UG) substrates using an in vitro kinase assay previously described . .. shRNA Suppression of Akt1 and p27 Akt1 and p27 shRNA oligonucleotides were designed against three different sequences within their respective target RNA ( ) and cloned into pLKO.1-TRC (Addgene plasmid 10878, ).

Kinase Assay:

Article Title: Cytoplasmic Localization of p21 Protects Trophoblast Giant Cells from DNA Damage Induced Apoptosis
Article Snippet: .. Protein Kinase Assays Recombinant mouse p21 proteins were purified from bacteria and tested as Akt1 (Sigma SRP-0353-10UG) substrates using an in vitro kinase assay previously described . .. shRNA Suppression of Akt1 and p27 Akt1 and p27 shRNA oligonucleotides were designed against three different sequences within their respective target RNA ( ) and cloned into pLKO.1-TRC (Addgene plasmid 10878, ).

Binding Assay:

Article Title: Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock *
Article Snippet: .. Pulldown Assay for Protein-Protein Interactions Purified recombinant proteins at 0.1 nm were mixed in 200 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 100 μg/ml of BSA, 0.05% Nonidet P-40) and incubated at 22 °C for 15 min. Then 20 μl of either FLAG- or V5-agarose (Sigma) beads were added and the mixture was incubated at 4 °C for 90 min. Then, the beads were washed with 1 ml of ice-cold binding buffer 4 times and after the final wash resuspended in 25 μl of SDS loading buffer and heated at 95 °C for 5 min. .. The proteins were then separated on SDS-PAGE and visualized by immunoblotting.

Transformation Assay:

Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
Article Snippet: .. Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Mass spectrometry analysis was performed on Coomassie-stained gel bands corresponding to the readthrough GST proteins.

Plasmid Preparation:

Article Title: Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Article Snippet: .. Expression and purification of labeled ComGC for NMR The DNA sequence of ComGC lacking the signal peptide and codons for the N-terminal hydrophobic domain (ComGCΔ1–39) was cloned downstream of the His6 tag sequence into pet28a vector (Novagen). .. Constructs were confirmed by sequencing and transformed into E. coli Rosetta (DE3).

Article Title: Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway
Article Snippet: .. Recombinant protein purification FLAG-EGFP and FLAG-EGFP-Tsg101-CTD (both with the A206K mutation in EGFP that renders it monomeric), as well as full length NiV-C (native sequence) with a N-terminal HA tag, were inserted into the pET-15b vector (Novagen), which appends an additional N-terminal 6XHis tag. .. The recombinant proteins were purified from BL21(DE3) E . coli as previously described [ ] with minor modifications.

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    Millipore pet28a fusion expression vector
    Structural design and construction of 3′ end PCC fusion subunits variants. (a) 3′ end fusion subunits variants were designed to be amplified from original PCC subunits clone. MTS-TAT fusion subunits could be amplified from MTS fusion subunits. (b) A schematic representation of TAT, MTS, and TAT-MTS fusion 5′ end subunits constructs. Control variants are subunits lacking the MTS or TAT domains. (c) PCR amplification results of pccA and pccB variants by using KOD DNA polymerase. (d) Digestion of cloned 3′ end fusion pccA and pccB subunits in <t>pET28a</t> vector by restriction enzymes. pccA-TAT, pccA-MTS and pccA-MTS-TAT clones were digested by Eco RI-HF and Hin dIII-HF restriction enzymes. pccB-TAT, pccB-MTS and pccB-MTS-TAT clones were digested by Not I-HF and Bam HI-HF restriction enzymes. All digestions were carried out in 37 °C overnight and digestion results visualized by GelRed staining on 10% agarose gel.
    Pet28a Fusion Expression Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pet28a fusion expression vector/product/Millipore
    Average 90 stars, based on 1 article reviews
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    pet28a fusion expression vector - by Bioz Stars, 2020-08
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    93
    Millipore plasmid pet28a
    Plasmid maps of pCNA, pSNK, and pBDC. The helper plasmids pCNA and pSNK, which could be eliminated by incubation at 42°C, were obtained upon insertion of I-CreI and I-SceI , respectively, into pKOBEG. The bla cassette was obtained from pET3b (Novagen), and the kan cassette was obtained from <t>pET28a</t> (Novagen). The p15A ori and cat cassettes of the landing pad plasmid pBDC were obtained from pACYCDuet-1 (commercial plasmid purchased from EMD Biosciences). IC and IS represent the I-CreI endonuclease recognition site and I-SceI endonuclease recognition site, respectively.
    Plasmid Pet28a, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 536 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pet28a/product/Millipore
    Average 93 stars, based on 536 article reviews
    Price from $9.99 to $1999.99
    plasmid pet28a - by Bioz Stars, 2020-08
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    Image Search Results


    Structural design and construction of 3′ end PCC fusion subunits variants. (a) 3′ end fusion subunits variants were designed to be amplified from original PCC subunits clone. MTS-TAT fusion subunits could be amplified from MTS fusion subunits. (b) A schematic representation of TAT, MTS, and TAT-MTS fusion 5′ end subunits constructs. Control variants are subunits lacking the MTS or TAT domains. (c) PCR amplification results of pccA and pccB variants by using KOD DNA polymerase. (d) Digestion of cloned 3′ end fusion pccA and pccB subunits in pET28a vector by restriction enzymes. pccA-TAT, pccA-MTS and pccA-MTS-TAT clones were digested by Eco RI-HF and Hin dIII-HF restriction enzymes. pccB-TAT, pccB-MTS and pccB-MTS-TAT clones were digested by Not I-HF and Bam HI-HF restriction enzymes. All digestions were carried out in 37 °C overnight and digestion results visualized by GelRed staining on 10% agarose gel.

    Journal: Molecular Genetics and Metabolism Reports

    Article Title: Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia

    doi: 10.1016/j.ymgmr.2016.06.009

    Figure Lengend Snippet: Structural design and construction of 3′ end PCC fusion subunits variants. (a) 3′ end fusion subunits variants were designed to be amplified from original PCC subunits clone. MTS-TAT fusion subunits could be amplified from MTS fusion subunits. (b) A schematic representation of TAT, MTS, and TAT-MTS fusion 5′ end subunits constructs. Control variants are subunits lacking the MTS or TAT domains. (c) PCR amplification results of pccA and pccB variants by using KOD DNA polymerase. (d) Digestion of cloned 3′ end fusion pccA and pccB subunits in pET28a vector by restriction enzymes. pccA-TAT, pccA-MTS and pccA-MTS-TAT clones were digested by Eco RI-HF and Hin dIII-HF restriction enzymes. pccB-TAT, pccB-MTS and pccB-MTS-TAT clones were digested by Not I-HF and Bam HI-HF restriction enzymes. All digestions were carried out in 37 °C overnight and digestion results visualized by GelRed staining on 10% agarose gel.

    Article Snippet: The PCR products for pccA and pccB fusion constructs were digested by Eco RI-HF/Hin dIII-HF and Bam HI-HF/Not I-HF endonucleases respectively before ligation into pET28a fusion expression vector (Merck Millipore Division, Merck Pte.

    Techniques: Periodic Counter-current Chromatography, Amplification, Construct, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Staining, Agarose Gel Electrophoresis

    Plasmid maps of pCNA, pSNK, and pBDC. The helper plasmids pCNA and pSNK, which could be eliminated by incubation at 42°C, were obtained upon insertion of I-CreI and I-SceI , respectively, into pKOBEG. The bla cassette was obtained from pET3b (Novagen), and the kan cassette was obtained from pET28a (Novagen). The p15A ori and cat cassettes of the landing pad plasmid pBDC were obtained from pACYCDuet-1 (commercial plasmid purchased from EMD Biosciences). IC and IS represent the I-CreI endonuclease recognition site and I-SceI endonuclease recognition site, respectively.

    Journal: PLoS ONE

    Article Title: An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome

    doi: 10.1371/journal.pone.0186891

    Figure Lengend Snippet: Plasmid maps of pCNA, pSNK, and pBDC. The helper plasmids pCNA and pSNK, which could be eliminated by incubation at 42°C, were obtained upon insertion of I-CreI and I-SceI , respectively, into pKOBEG. The bla cassette was obtained from pET3b (Novagen), and the kan cassette was obtained from pET28a (Novagen). The p15A ori and cat cassettes of the landing pad plasmid pBDC were obtained from pACYCDuet-1 (commercial plasmid purchased from EMD Biosciences). IC and IS represent the I-CreI endonuclease recognition site and I-SceI endonuclease recognition site, respectively.

    Article Snippet: The commercial plasmid pET28a was purchased from EMD Biosciences (Novagen).

    Techniques: Plasmid Preparation, Incubation

    Expression and isolation of recombinant FopB protein. Total protein was prepared from pET28a-FopB-transformed E. coli BL21(DE3) cells or from the pET28a vector with or without IPTG induction and was visualized by Coomassie blue staining. Also shown are

    Journal:

    Article Title: Francisella tularensis T-Cell Antigen Identification Using Humanized HLA-DR4 Transgenic Mice ▿

    doi: 10.1128/CVI.00361-09

    Figure Lengend Snippet: Expression and isolation of recombinant FopB protein. Total protein was prepared from pET28a-FopB-transformed E. coli BL21(DE3) cells or from the pET28a vector with or without IPTG induction and was visualized by Coomassie blue staining. Also shown are

    Article Snippet: The 0.54-kb fopB digest was inserted into the pET28a expression vector, using the same restriction sites, and the resulting plasmid (pET28-FopB) was used to transform E. coli BL21(DE3) (EMD Biosciences, Gibbstown, NJ) for protein expression.

    Techniques: Expressing, Isolation, Recombinant, Transformation Assay, Plasmid Preparation, Staining

    Vip3A protein purified after vip3A expression in E. coli . (A) pET28a + expression vector containing synthetic vip3A gene, spliced with poly-histidine tag for specific protein purification, under T7 promoter and terminator, (B) Coomassie blue stained SDS-PAGE gel analysis for recombinant insecticidal vip3A protein expressed by E. coli BL21 (DC3) using pET expression system. (C) Western blot analysis of purified Vip3A protein, using monoclonal antipoly histidine primary antibodies.

    Journal: bioRxiv

    Article Title: Comparison of in vitro and in planta toxicity of Vip3A for lepidopteran herbivores

    doi: 10.1101/829895

    Figure Lengend Snippet: Vip3A protein purified after vip3A expression in E. coli . (A) pET28a + expression vector containing synthetic vip3A gene, spliced with poly-histidine tag for specific protein purification, under T7 promoter and terminator, (B) Coomassie blue stained SDS-PAGE gel analysis for recombinant insecticidal vip3A protein expressed by E. coli BL21 (DC3) using pET expression system. (C) Western blot analysis of purified Vip3A protein, using monoclonal antipoly histidine primary antibodies.

    Article Snippet: The PCR product cloned in the corresponding restriction sites of the pET28a+ vector (EMD Biosciences, San Diego, CA).

    Techniques: Purification, Expressing, Plasmid Preparation, Protein Purification, Staining, SDS Page, Recombinant, Positron Emission Tomography, Western Blot