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Agilent technologies e coli bl21
E Coli Bl21, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens
Article Snippet: .. Several different Escherichia coli strains were used in this study: E. coli XL1-Blue MRF′ (Agilent Technologies, Santa Clara, CA) for antibacterial activity testing, E. coli TOP10 (Life Technologies, Carlsbad, CA) for DNA cloning and cell stock storage, and E. coli BL21(DE3) pLysS, BL21-CodonPlus-(DE3)-RIL, and BL21-CodonPlus-(DE3)-RP (Agilent Technologies) as host strains for protein expression. .. For proper selection, ampicillin (Roche Diagnostics, Mannheim, Germany) (100 µg/ml) or chloramphenicol (Calbiochem, Darmstadt, Germany) (50 µg/ml) was used.

Construct:

Article Title: HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C
Article Snippet: .. Expression and purification of HspB1 constructs A pET28d(+) vector encoding human HspB1 ACD (residues 84 to 171) was transformed into Escherichia coli BL21(DE3) cells (Agilent) and expressed and purified as described previously ( ). .. For crystallography, residue K171 was deleted using a site-directed mutagenesis kit (Agilent) and HspB184–170 was expressed and purified as described.

Article Title: SETD3 protein is the actin-specific histidine N-methyltransferase
Article Snippet: .. For β-actin production, E. coli BL21(DE3) (Agilent, USA) cells were transformed with the DNA construct and a single colony was selected to start an over-night pre-culture. .. 500 mL of LB broth (with 100 μg/mL ampicilin) was inoculated with 50 ml of the pre-culture and incubated at 37°C and 200 rpm until an OD600 of 0.5 was reached.

Purification:

Article Title: HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C
Article Snippet: .. Expression and purification of HspB1 constructs A pET28d(+) vector encoding human HspB1 ACD (residues 84 to 171) was transformed into Escherichia coli BL21(DE3) cells (Agilent) and expressed and purified as described previously ( ). .. For crystallography, residue K171 was deleted using a site-directed mutagenesis kit (Agilent) and HspB184–170 was expressed and purified as described.

Article Title: A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner
Article Snippet: .. Recombinant proteins for TFIIAα/β, TFIIAγ, TBP, and GAL4–VP16 were expressed with Escherichia coli BL21 (DE3; Agilent) and purified with nickel-agarose as described in the manufacturer's manual (Qiagen). .. Reconstituted TFIIA was obtained through denaturing the recombinant proteins TFIIAα/β and TFIIAγ with protein denature buffer (50 m m Tris-Cl, pH 7.4,, 1 m m EDTA, 100 m m NaCl, 0.1 m m PMSF, and 7 m urea) for 4 h, and then renaturing the proteins by dialysis (20 m m HEPES, pH 7.6, 1 m m EDTA, 100 m m NaCl, 0.1 m m PMSF, 10% glycerol, and 0–7 m urea); the concentration of urea in dialyzing buffer was decreased at 1 m per 5 h from 7 to 0 m during the dialysis.

Activity Assay:

Article Title: Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens
Article Snippet: .. Several different Escherichia coli strains were used in this study: E. coli XL1-Blue MRF′ (Agilent Technologies, Santa Clara, CA) for antibacterial activity testing, E. coli TOP10 (Life Technologies, Carlsbad, CA) for DNA cloning and cell stock storage, and E. coli BL21(DE3) pLysS, BL21-CodonPlus-(DE3)-RIL, and BL21-CodonPlus-(DE3)-RP (Agilent Technologies) as host strains for protein expression. .. For proper selection, ampicillin (Roche Diagnostics, Mannheim, Germany) (100 µg/ml) or chloramphenicol (Calbiochem, Darmstadt, Germany) (50 µg/ml) was used.

Expressing:

Article Title: HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C
Article Snippet: .. Expression and purification of HspB1 constructs A pET28d(+) vector encoding human HspB1 ACD (residues 84 to 171) was transformed into Escherichia coli BL21(DE3) cells (Agilent) and expressed and purified as described previously ( ). .. For crystallography, residue K171 was deleted using a site-directed mutagenesis kit (Agilent) and HspB184–170 was expressed and purified as described.

Article Title: HTLV-1 HBZ PROTEIN DEREGULATES INTERACTIONS BETWEEN CELLULAR FACTORS AND THE KIX DOMAIN OF p300/CBP
Article Snippet: .. E. coli BL21(DE3) pLysS cells (Agilent Technologies) were used for expression of Gal4-HBZ-AD wt and mutants, HBZ-AD wt and mutants, HA-c-Myb-AD, GST-CREB, GST-KIX, untagged KIX and CREB. .. BL21-codon plus (DE3) cells (Agilent Technologies) were used for expression of Gal4-HBZ, Flag-MLL-AD and GST-HBZ.

Article Title: Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens
Article Snippet: .. Several different Escherichia coli strains were used in this study: E. coli XL1-Blue MRF′ (Agilent Technologies, Santa Clara, CA) for antibacterial activity testing, E. coli TOP10 (Life Technologies, Carlsbad, CA) for DNA cloning and cell stock storage, and E. coli BL21(DE3) pLysS, BL21-CodonPlus-(DE3)-RIL, and BL21-CodonPlus-(DE3)-RP (Agilent Technologies) as host strains for protein expression. .. For proper selection, ampicillin (Roche Diagnostics, Mannheim, Germany) (100 µg/ml) or chloramphenicol (Calbiochem, Darmstadt, Germany) (50 µg/ml) was used.

Article Title: Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73
Article Snippet: .. These recombinant vectors were transformed into E. coli BL21 (DE3; Agilent Technologies, Santa Clara, CA, USA) for protein expression. .. Positive clones bearing the gene were identified by PCR after culturing cells in Luria-Bertani (LB) medium containing ampicillin with shaking (200 rpm) at 37°C.

Transformation Assay:

Article Title: Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth
Article Snippet: .. In brief, Escherichia coli BL21(DE3)RIPL (Agilent Technologies) cells transformed with vinculin plasmids were grown in Lysogeny broth-rich media at 37°C to an A 600 of 0.6. .. The cultures were cooled to 18°C before adding isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.5 mM to induce vinculin expression overnight.

Article Title: HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C
Article Snippet: .. Expression and purification of HspB1 constructs A pET28d(+) vector encoding human HspB1 ACD (residues 84 to 171) was transformed into Escherichia coli BL21(DE3) cells (Agilent) and expressed and purified as described previously ( ). .. For crystallography, residue K171 was deleted using a site-directed mutagenesis kit (Agilent) and HspB184–170 was expressed and purified as described.

Article Title: SETD3 protein is the actin-specific histidine N-methyltransferase
Article Snippet: .. For β-actin production, E. coli BL21(DE3) (Agilent, USA) cells were transformed with the DNA construct and a single colony was selected to start an over-night pre-culture. .. 500 mL of LB broth (with 100 μg/mL ampicilin) was inoculated with 50 ml of the pre-culture and incubated at 37°C and 200 rpm until an OD600 of 0.5 was reached.

Article Title: Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73
Article Snippet: .. These recombinant vectors were transformed into E. coli BL21 (DE3; Agilent Technologies, Santa Clara, CA, USA) for protein expression. .. Positive clones bearing the gene were identified by PCR after culturing cells in Luria-Bertani (LB) medium containing ampicillin with shaking (200 rpm) at 37°C.

Recombinant:

Article Title: A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner
Article Snippet: .. Recombinant proteins for TFIIAα/β, TFIIAγ, TBP, and GAL4–VP16 were expressed with Escherichia coli BL21 (DE3; Agilent) and purified with nickel-agarose as described in the manufacturer's manual (Qiagen). .. Reconstituted TFIIA was obtained through denaturing the recombinant proteins TFIIAα/β and TFIIAγ with protein denature buffer (50 m m Tris-Cl, pH 7.4,, 1 m m EDTA, 100 m m NaCl, 0.1 m m PMSF, and 7 m urea) for 4 h, and then renaturing the proteins by dialysis (20 m m HEPES, pH 7.6, 1 m m EDTA, 100 m m NaCl, 0.1 m m PMSF, 10% glycerol, and 0–7 m urea); the concentration of urea in dialyzing buffer was decreased at 1 m per 5 h from 7 to 0 m during the dialysis.

Article Title: Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73
Article Snippet: .. These recombinant vectors were transformed into E. coli BL21 (DE3; Agilent Technologies, Santa Clara, CA, USA) for protein expression. .. Positive clones bearing the gene were identified by PCR after culturing cells in Luria-Bertani (LB) medium containing ampicillin with shaking (200 rpm) at 37°C.

Plasmid Preparation:

Article Title: HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C
Article Snippet: .. Expression and purification of HspB1 constructs A pET28d(+) vector encoding human HspB1 ACD (residues 84 to 171) was transformed into Escherichia coli BL21(DE3) cells (Agilent) and expressed and purified as described previously ( ). .. For crystallography, residue K171 was deleted using a site-directed mutagenesis kit (Agilent) and HspB184–170 was expressed and purified as described.

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  • 91
    Agilent technologies bl21 e coli
    Adseverin D5 is not is expressed during  in vitro  OCG. A ) PCR detected only the full-length Ads isoform in Day 0 and Day 6 BMMs and Day 0 and Day 4 RAW macrophage cultures. pSE380-Ads (full-length Ads) and pSE380-Ads-D5 (Ads D5 isoform) plasmids were used as positive controls. Equal volumes of cDNA from each representative samples were used as templates. The intensity of the signal is not a quantitative measure of Ads gene expression.  B ) Immunoblot analysis also failed to detect the Ads D5 in BMM and RAW macrophage-derived osteoclasts. Lysates from the transformed BL21  E. coli  induced to express exogenous protein were used as positive controls. Only the 85 kDa full-length isoform was detected in 20 µg of TCL from Day 6 BMMs and Day 4 RAW macrophages cultures. Ads was not detected in 20 µg of TCL from unstimulated samples.
    Bl21 E Coli, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies bl21 cp
    Screening for soluble Salp15 HBc fusions. Cells from induced cultures of <t>BL21*CP</t> cells transformed with the indicated tick protein—HBc fusion constructs were lysed under non-denaturing conditions. Unfractionated total lysate (T) and soluble supernatant (S) after centrifugation were compared by SDS-PAGE. For all SplitCore constructs, the gel section with the tick protein containing large core segment is shown. wt, Wild-type protein; C - , Cys-free variant. (A) Contiguous chain HBc149 with c/e1 inserted Salp15. The complete gel lanes are shown inS 3A Fig. (B) SplitCore with Salp15 fused to coreN. (C) SplitCore constructs with coreN and coreC ORFs in reverse order (SplitCore REV). Failure of alternative expression strains to promote higher solubility of the wt-Salp15 fusion is shown in S3B Fig . (D) SplitCore REV constructs bearing Salp15 on both coreN and coreC. (E) SplitCore REV construct bearing Cys-free Iric-1 on coreN. The complete gel lanes are shown in S3C Fig . Note the strong positive impact on solubility of replacing the endogenous tick protein cysteines in the SplitCore REV but not the contiguous chain HBc constructs.
    Bl21 Cp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies bl21 gold cells
    —Passaging scheme over course of experiment. Four replicate T7Δ1 phage lines were initially subjected to 50 passages on <t>BL21</t> cells expressing the G78-KIRV RNAP mutant using 100 µM IPTG in trans (i.e., from a plasmid). Passaging was continued for another 50 passages in the same manner using 0 µM IPTG induction.
    Bl21 Gold Cells, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies bl21 de3 codonplus ril
    Enzymatic activity assays using glucanase- and xylanase-IFP fusion proteins expressed in E. coli . LIC-pDEST-LC1-/LC2 vectors encoding 6xHis-IFP-TEV-endo-β-1,4-glucanase/-xylanase and endo-β-1,4-glucanase-/xylanase-TEV-IFP-6xHis fusion proteins were transformed into the E. coli strains <t>BL21</t> (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) <t>CodonPlus-RIL</t> (‘Codon’) and Rosetta (DE3) pRARE (‘Rosetta’). Enzymatic activity was tested by Congo Red staining and destaining with 1 M NaCl on carboxymethylcellulose- (left panel) or xylan- (right panel) containing agar plates after transferring 2 µL of the respective expression strains and over-night incubation at 37°C. Glucanase activity leads to the formation of a white halo around the colonies, whereas xylanase activity leads to the formation of a black halo [31] . E. coli cells expressing IFP-6xHis fusion protein were used as negative control, and cell-free supernatant of Pichia pastoris expression cultures containing secreted endo-β-1,4-glucanase-myc-6xHis or endo-β-1,4-xylanase-myc-6xHis fusion proteins were used as positive controls (P).
    Bl21 De3 Codonplus Ril, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Adseverin D5 is not is expressed during  in vitro  OCG. A ) PCR detected only the full-length Ads isoform in Day 0 and Day 6 BMMs and Day 0 and Day 4 RAW macrophage cultures. pSE380-Ads (full-length Ads) and pSE380-Ads-D5 (Ads D5 isoform) plasmids were used as positive controls. Equal volumes of cDNA from each representative samples were used as templates. The intensity of the signal is not a quantitative measure of Ads gene expression.  B ) Immunoblot analysis also failed to detect the Ads D5 in BMM and RAW macrophage-derived osteoclasts. Lysates from the transformed BL21  E. coli  induced to express exogenous protein were used as positive controls. Only the 85 kDa full-length isoform was detected in 20 µg of TCL from Day 6 BMMs and Day 4 RAW macrophages cultures. Ads was not detected in 20 µg of TCL from unstimulated samples.

    Journal: PLoS ONE

    Article Title: The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    doi: 10.1371/journal.pone.0109078

    Figure Lengend Snippet: Adseverin D5 is not is expressed during in vitro OCG. A ) PCR detected only the full-length Ads isoform in Day 0 and Day 6 BMMs and Day 0 and Day 4 RAW macrophage cultures. pSE380-Ads (full-length Ads) and pSE380-Ads-D5 (Ads D5 isoform) plasmids were used as positive controls. Equal volumes of cDNA from each representative samples were used as templates. The intensity of the signal is not a quantitative measure of Ads gene expression. B ) Immunoblot analysis also failed to detect the Ads D5 in BMM and RAW macrophage-derived osteoclasts. Lysates from the transformed BL21 E. coli induced to express exogenous protein were used as positive controls. Only the 85 kDa full-length isoform was detected in 20 µg of TCL from Day 6 BMMs and Day 4 RAW macrophages cultures. Ads was not detected in 20 µg of TCL from unstimulated samples.

    Article Snippet: Two distinct bands at 85 kDa and 80 kDa are readily identifiable in lysates of BL21 E. coli induced to express exogenous Ads or Ads D5, respectively.

    Techniques: In Vitro, Polymerase Chain Reaction, Expressing, Derivative Assay, Transformation Assay

    Screening for soluble Salp15 HBc fusions. Cells from induced cultures of BL21*CP cells transformed with the indicated tick protein—HBc fusion constructs were lysed under non-denaturing conditions. Unfractionated total lysate (T) and soluble supernatant (S) after centrifugation were compared by SDS-PAGE. For all SplitCore constructs, the gel section with the tick protein containing large core segment is shown. wt, Wild-type protein; C - , Cys-free variant. (A) Contiguous chain HBc149 with c/e1 inserted Salp15. The complete gel lanes are shown inS 3A Fig. (B) SplitCore with Salp15 fused to coreN. (C) SplitCore constructs with coreN and coreC ORFs in reverse order (SplitCore REV). Failure of alternative expression strains to promote higher solubility of the wt-Salp15 fusion is shown in S3B Fig . (D) SplitCore REV constructs bearing Salp15 on both coreN and coreC. (E) SplitCore REV construct bearing Cys-free Iric-1 on coreN. The complete gel lanes are shown in S3C Fig . Note the strong positive impact on solubility of replacing the endogenous tick protein cysteines in the SplitCore REV but not the contiguous chain HBc constructs.

    Journal: PLoS ONE

    Article Title: Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

    doi: 10.1371/journal.pone.0136180

    Figure Lengend Snippet: Screening for soluble Salp15 HBc fusions. Cells from induced cultures of BL21*CP cells transformed with the indicated tick protein—HBc fusion constructs were lysed under non-denaturing conditions. Unfractionated total lysate (T) and soluble supernatant (S) after centrifugation were compared by SDS-PAGE. For all SplitCore constructs, the gel section with the tick protein containing large core segment is shown. wt, Wild-type protein; C - , Cys-free variant. (A) Contiguous chain HBc149 with c/e1 inserted Salp15. The complete gel lanes are shown inS 3A Fig. (B) SplitCore with Salp15 fused to coreN. (C) SplitCore constructs with coreN and coreC ORFs in reverse order (SplitCore REV). Failure of alternative expression strains to promote higher solubility of the wt-Salp15 fusion is shown in S3B Fig . (D) SplitCore REV constructs bearing Salp15 on both coreN and coreC. (E) SplitCore REV construct bearing Cys-free Iric-1 on coreN. The complete gel lanes are shown in S3C Fig . Note the strong positive impact on solubility of replacing the endogenous tick protein cysteines in the SplitCore REV but not the contiguous chain HBc constructs.

    Article Snippet: Induction was performed, as usual, for 12–15 h at 22°C in BL21*CP and T7 SHuffle Express CP cells, or at 15°C in Arctic Express cells (Agilent).

    Techniques: Transformation Assay, Construct, Centrifugation, SDS Page, Variant Assay, Expressing, Solubility

    Expression in E . coli of tHRF yields soluble monomeric protein compatible with contiguous chain c/e1 insertion into HBc. (A) N terminally His6 tagged tHRF (H6-tHRF). Left : SDS-PAGE analysis of native lysate of transformed BL21*CP cells. T, total unfractionated lysate; P and S, insoluble pellet and soluble supernatant after centrifugation. The gel was stained with Coomassie Blue. Middle : Ni 2+ IMAC of the soluble lysate fraction. Equal aliquots from the IMAC fractions eluted by increasing imidazole concentration were analyzed by SDS-PAGE. Right : Size exclusion chromatography (SEC). Peak fractions from IMAC were subjected to SEC on a Superdex S75 16/60 column; elution volumes are indicated on the top. By comparison with the elution volumes of marker proteins the peak of tHRF eluted at a volume corresponding to a molecular mass of ~27 kDa (arrowhead). This was confirmed for a non-tagged version of recombinant tHRF ( S1 Fig ). (B) HBc149_c/e1-tHRF. Left : Cleared native lysate of bacteria expressing the contiguous chain fusion protein were subjected to sucrose gradient sedimentation. Equal aliquots from fractions 1 to 13 (out of 14 total) were analyzed by SDS-PAGE. Middle : Immunoblot after native agarose gel electrophoresis (NAGE). Material from fraction 8 of the gradients shown in (B) and (C) was subjected to NAGE, blotted and detected using the HBc particle-specific mab mc275. Right : Negative staining EM. (B) HBc183_c/e1-tHRF. Sedimentation and EM analysis were performed as in (B).

    Journal: PLoS ONE

    Article Title: Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

    doi: 10.1371/journal.pone.0136180

    Figure Lengend Snippet: Expression in E . coli of tHRF yields soluble monomeric protein compatible with contiguous chain c/e1 insertion into HBc. (A) N terminally His6 tagged tHRF (H6-tHRF). Left : SDS-PAGE analysis of native lysate of transformed BL21*CP cells. T, total unfractionated lysate; P and S, insoluble pellet and soluble supernatant after centrifugation. The gel was stained with Coomassie Blue. Middle : Ni 2+ IMAC of the soluble lysate fraction. Equal aliquots from the IMAC fractions eluted by increasing imidazole concentration were analyzed by SDS-PAGE. Right : Size exclusion chromatography (SEC). Peak fractions from IMAC were subjected to SEC on a Superdex S75 16/60 column; elution volumes are indicated on the top. By comparison with the elution volumes of marker proteins the peak of tHRF eluted at a volume corresponding to a molecular mass of ~27 kDa (arrowhead). This was confirmed for a non-tagged version of recombinant tHRF ( S1 Fig ). (B) HBc149_c/e1-tHRF. Left : Cleared native lysate of bacteria expressing the contiguous chain fusion protein were subjected to sucrose gradient sedimentation. Equal aliquots from fractions 1 to 13 (out of 14 total) were analyzed by SDS-PAGE. Middle : Immunoblot after native agarose gel electrophoresis (NAGE). Material from fraction 8 of the gradients shown in (B) and (C) was subjected to NAGE, blotted and detected using the HBc particle-specific mab mc275. Right : Negative staining EM. (B) HBc183_c/e1-tHRF. Sedimentation and EM analysis were performed as in (B).

    Article Snippet: Induction was performed, as usual, for 12–15 h at 22°C in BL21*CP and T7 SHuffle Express CP cells, or at 15°C in Arctic Express cells (Agilent).

    Techniques: Expressing, SDS Page, Transformation Assay, Centrifugation, Staining, Concentration Assay, Size-exclusion Chromatography, Marker, Recombinant, Sedimentation, Agarose Gel Electrophoresis, Negative Staining

    —Passaging scheme over course of experiment. Four replicate T7Δ1 phage lines were initially subjected to 50 passages on BL21 cells expressing the G78-KIRV RNAP mutant using 100 µM IPTG in trans (i.e., from a plasmid). Passaging was continued for another 50 passages in the same manner using 0 µM IPTG induction.

    Journal: Genome Biology and Evolution

    Article Title: Predicting Evolution of the Transcription Regulatory Network in a Bacteriophage

    doi: 10.1093/gbe/evy191

    Figure Lengend Snippet: —Passaging scheme over course of experiment. Four replicate T7Δ1 phage lines were initially subjected to 50 passages on BL21 cells expressing the G78-KIRV RNAP mutant using 100 µM IPTG in trans (i.e., from a plasmid). Passaging was continued for another 50 passages in the same manner using 0 µM IPTG induction.

    Article Snippet: Phage Passaging Frozen aliquots of BL21-Gold cells (Agilent) transformed with pLUV-G78-KIRV RNAP (wild-type T7 RNAP with Q744K, L747V, N748H, L749I, R756E, L757M, H772R, and E775V mutations, from ) were thawed and used to inoculate 10 ml of 2xYT with proper antibiotic and grown at 37 °C to achieve a density of 108 cells/ml at 60 min. For the first 50 passages, isopropyl-l -thio-β-galactoside (IPTG) was added to a concentration of 1 mM before the 60 min of growth (no IPTG addition for the second 50 passages).

    Techniques: Passaging, Expressing, Mutagenesis, Plasmid Preparation

    Enzymatic activity assays using glucanase- and xylanase-IFP fusion proteins expressed in E. coli . LIC-pDEST-LC1-/LC2 vectors encoding 6xHis-IFP-TEV-endo-β-1,4-glucanase/-xylanase and endo-β-1,4-glucanase-/xylanase-TEV-IFP-6xHis fusion proteins were transformed into the E. coli strains BL21 (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) CodonPlus-RIL (‘Codon’) and Rosetta (DE3) pRARE (‘Rosetta’). Enzymatic activity was tested by Congo Red staining and destaining with 1 M NaCl on carboxymethylcellulose- (left panel) or xylan- (right panel) containing agar plates after transferring 2 µL of the respective expression strains and over-night incubation at 37°C. Glucanase activity leads to the formation of a white halo around the colonies, whereas xylanase activity leads to the formation of a black halo [31] . E. coli cells expressing IFP-6xHis fusion protein were used as negative control, and cell-free supernatant of Pichia pastoris expression cultures containing secreted endo-β-1,4-glucanase-myc-6xHis or endo-β-1,4-xylanase-myc-6xHis fusion proteins were used as positive controls (P).

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Enzymatic activity assays using glucanase- and xylanase-IFP fusion proteins expressed in E. coli . LIC-pDEST-LC1-/LC2 vectors encoding 6xHis-IFP-TEV-endo-β-1,4-glucanase/-xylanase and endo-β-1,4-glucanase-/xylanase-TEV-IFP-6xHis fusion proteins were transformed into the E. coli strains BL21 (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) CodonPlus-RIL (‘Codon’) and Rosetta (DE3) pRARE (‘Rosetta’). Enzymatic activity was tested by Congo Red staining and destaining with 1 M NaCl on carboxymethylcellulose- (left panel) or xylan- (right panel) containing agar plates after transferring 2 µL of the respective expression strains and over-night incubation at 37°C. Glucanase activity leads to the formation of a white halo around the colonies, whereas xylanase activity leads to the formation of a black halo [31] . E. coli cells expressing IFP-6xHis fusion protein were used as negative control, and cell-free supernatant of Pichia pastoris expression cultures containing secreted endo-β-1,4-glucanase-myc-6xHis or endo-β-1,4-xylanase-myc-6xHis fusion proteins were used as positive controls (P).

    Article Snippet: Protein expression in E. coli and TEV protease cleavage For protein expression in E. coli LIC-pDEST-LC1/LC2 plasmid templates were transformed into different expression strains, i.e. BL21 (DE3) pLysS (Agilent Technologies, Waldbronn, Germany), BL21 (DE3) CodonPlus-RIL (Agilent Technologies), and Rosetta (DE3) pRARE (Merck, Darmstadt, Germany).

    Techniques: Activity Assay, Transformation Assay, Staining, Transferring, Expressing, Incubation, Negative Control

    Infrared analysis of in vitro and in vivo expressed IFP fusion proteins. Infrared scanning of all samples was performed in microtiter plates using the Odyssey Infrared Imaging System from LI-COR Biosciences. ( A ) In vitro transcription/translation products were analysed by infrared scanning using the whole reaction mixtures. 6xHis-GFP and IFP-6xHis fusion protein-expressing samples were used as negative (-) and positive (+) controls, respectively. ( B ) In-cell detection of IFP fusion protein (6xHis-IFP-TEV-ANAC042 shown as an example) in two randomly selected clones each from the E. coli strains (BL21 (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) CodonPlus-RIL (‘Codon’), and Rosetta (DE3) pRARE (‘Rosetta’). 6xHis-GFP and IFP-6xHis fusion protein-expressing cells were used as negative (-) and positive (+) controls, respectively. ( C ), ( D ) and ( E ) In-cell detection of IFP fusion protein (SAM1-TEV-IFP-6xHis shown as an example) in randomly selected K. lactis , P. pastoris and L. tarentolae clones. Cell lines not expressing IPF or expressing IFP-6xHis fusion protein were used as negative (-) and positive (+) controls, respectively. No positive control was available for expression in K. lactis . Note that strong infrared signal appears white in the digital images.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Infrared analysis of in vitro and in vivo expressed IFP fusion proteins. Infrared scanning of all samples was performed in microtiter plates using the Odyssey Infrared Imaging System from LI-COR Biosciences. ( A ) In vitro transcription/translation products were analysed by infrared scanning using the whole reaction mixtures. 6xHis-GFP and IFP-6xHis fusion protein-expressing samples were used as negative (-) and positive (+) controls, respectively. ( B ) In-cell detection of IFP fusion protein (6xHis-IFP-TEV-ANAC042 shown as an example) in two randomly selected clones each from the E. coli strains (BL21 (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) CodonPlus-RIL (‘Codon’), and Rosetta (DE3) pRARE (‘Rosetta’). 6xHis-GFP and IFP-6xHis fusion protein-expressing cells were used as negative (-) and positive (+) controls, respectively. ( C ), ( D ) and ( E ) In-cell detection of IFP fusion protein (SAM1-TEV-IFP-6xHis shown as an example) in randomly selected K. lactis , P. pastoris and L. tarentolae clones. Cell lines not expressing IPF or expressing IFP-6xHis fusion protein were used as negative (-) and positive (+) controls, respectively. No positive control was available for expression in K. lactis . Note that strong infrared signal appears white in the digital images.

    Article Snippet: Protein expression in E. coli and TEV protease cleavage For protein expression in E. coli LIC-pDEST-LC1/LC2 plasmid templates were transformed into different expression strains, i.e. BL21 (DE3) pLysS (Agilent Technologies, Waldbronn, Germany), BL21 (DE3) CodonPlus-RIL (Agilent Technologies), and Rosetta (DE3) pRARE (Merck, Darmstadt, Germany).

    Techniques: In Vitro, In Vivo, Imaging, Expressing, Clone Assay, Positive Control

    Expression of IFP fusion proteins in E. coli . Protein extracts obtained from IFP fusion protein-expressing E. coli strains BL21 (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) CodonPlus-RIL (‘Codon’), and Rosetta (DE3) pRARE (‘Rosetta’) were separated by SDS-PAGE and analysed by in-gel infrared imaging at 700 nm to detect IFP moieties (upper panel), followed by western transfer and immunological detection at 800 nm (using monoclonal mouse antibody directed against the 6xHis epitope; lower panel). IFP-6xHis fusion protein-expressing cells were used as positive control. Supernatant (S) and pellet (P) fractions of disrupted cells were analyzed after ultracentrifugation. M, molecular mass marker (kDa). Arrows indicate expected proteins.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Expression of IFP fusion proteins in E. coli . Protein extracts obtained from IFP fusion protein-expressing E. coli strains BL21 (DE3) pLysS (‘pLysS’), BL21 Star (DE3) pRARE (‘Star’), BL21 (DE3) CodonPlus-RIL (‘Codon’), and Rosetta (DE3) pRARE (‘Rosetta’) were separated by SDS-PAGE and analysed by in-gel infrared imaging at 700 nm to detect IFP moieties (upper panel), followed by western transfer and immunological detection at 800 nm (using monoclonal mouse antibody directed against the 6xHis epitope; lower panel). IFP-6xHis fusion protein-expressing cells were used as positive control. Supernatant (S) and pellet (P) fractions of disrupted cells were analyzed after ultracentrifugation. M, molecular mass marker (kDa). Arrows indicate expected proteins.

    Article Snippet: Protein expression in E. coli and TEV protease cleavage For protein expression in E. coli LIC-pDEST-LC1/LC2 plasmid templates were transformed into different expression strains, i.e. BL21 (DE3) pLysS (Agilent Technologies, Waldbronn, Germany), BL21 (DE3) CodonPlus-RIL (Agilent Technologies), and Rosetta (DE3) pRARE (Merck, Darmstadt, Germany).

    Techniques: Expressing, SDS Page, Imaging, Western Blot, Positive Control, Marker