bl21 Millipore Search Results


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  • 99
    Millipore e coli bl 21
    Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli <t>BL21/pET-21b-IFN-CSP</t> before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.
    E Coli Bl 21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore bl21 de3 millipore sigma
    Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli <t>BL21/pET-21b-IFN-CSP</t> before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.
    Bl21 De3 Millipore Sigma, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bl21 de3 competent cells
    Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli <t>BL21/pET-21b-IFN-CSP</t> before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.
    Bl21 De3 Competent Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bl21 de3 plyss
    Expression of UspF protein by an IPTG‐inducible E. coli <t>BL21</t> (DE3) <t>pLyS</t> strain and purification of soluble fractions containing UspF protein. A – T0 total protein extract before induction; T2, total protein extract after induction (3 h); T3, total protein extract after induction (16–18 h). The arrow indicates position of the UspF protein (18.4 kDa). B – Purification of soluble fractions or UspF protein by affinity chromatography using a nickel‐containing resin. 1–Flow through; 2–30 m M , 3–50 m M , 4–100 m M ; 5–200 m M of imidazole.
    Bl21 De3 Plyss, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore strain bl21
    SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli <t>BL21</t> . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively
    Strain Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore escherichiacoli bl21
    SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli <t>BL21</t> . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively
    Escherichiacoli Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore rosetta bl21
    SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli <t>BL21</t> . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively
    Rosetta Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA bl21
    SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli <t>BL21</t> . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively
    Bl21, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore bl21 plyss
    SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli <t>BL21</t> . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively
    Bl21 Plyss, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore escherichia coli
    Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, <t>Escherichia</t> coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.
    Escherichia Coli, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11586 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore bl21 de3 ril
    Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, <t>Escherichia</t> coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.
    Bl21 De3 Ril, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore bl21 λde3 plyse
    Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, <t>Escherichia</t> coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.
    Bl21 λde3 Plyse, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore competent bl21 de3
    Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, <t>Escherichia</t> coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.
    Competent Bl21 De3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore bl21 de3 r3 rosetta
    Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, <t>Escherichia</t> coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.
    Bl21 De3 R3 Rosetta, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bl21 de3 rosetta
    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
    Bl21 De3 Rosetta, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore bl21 de3 trxb
    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    95
    Millipore bl21 rosetta 2
    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
    Bl21 Rosetta 2, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore bacterial bl21 de3
    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
    Bacterial Bl21 De3, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore protein purification
    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
    Protein Purification, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bl21 bacteria
    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, <t>BL21</t> Gold, BL21 Codon (+) and BL21 <t>Rosetta</t> in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.
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    Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli <t>BL21</t> (DE3) <t>pLysS</t> <t>Rosetta</t> whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.
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    Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli <t>BL21</t> (DE3) <t>pLysS</t> <t>Rosetta</t> whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.
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    Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli <t>BL21</t> (DE3) <t>pLysS</t> <t>Rosetta</t> whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.
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    Image Search Results


    Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.

    Journal: BioMed Research International

    Article Title: Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of a Plasmodium Region I-Plus Peptide

    doi: 10.1155/2014/261631

    Figure Lengend Snippet: Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.

    Article Snippet: Bacterial Strains, Plasmids, and Culture Media E. coli strain DH5α (Novagen, USA) and BL21 (DE3; Novagen, USA) were used as the host for gene manipulation and expression of fusion protein, respectively. pMD20-T (Takara, Japan) and pET-21b (Novagen, USA) were applied for gene cloning and expression, respectively.

    Techniques: SDS Page, Western Blot, Expressing, Purification, Molecular Weight, Marker, Positron Emission Tomography, Centrifugation, Affinity Chromatography

    Expression analysis of recombinant ADAMTS1 in E. coli BL21 (DE3). ( A ) SDS-PAGE analysis of recombinant ADAMTS1 induced by IPTG. Lane 1: uninduced bacteria lysate; lane 2: IPTG wholly induced bacteria lysate; lane 3: supernatant of bacteria lysate; lane 4: precipitation of bacteria lysate; ( B ) SDS-PAGE analysis of purified fusion ADAMTS1 and ADAMTS1 on the Coomassie brilliant blue-stained gel; ( C ) Western blot analysis of purified fusion ADAMTS1 and ADAMTS1. Lane 1: purified fusion protein with NTA column; lane 2: the final purified protein after removal of thioredoxin using heparin-sepharose column.

    Journal: Molecules

    Article Title: Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

    doi: 10.3390/molecules161210709

    Figure Lengend Snippet: Expression analysis of recombinant ADAMTS1 in E. coli BL21 (DE3). ( A ) SDS-PAGE analysis of recombinant ADAMTS1 induced by IPTG. Lane 1: uninduced bacteria lysate; lane 2: IPTG wholly induced bacteria lysate; lane 3: supernatant of bacteria lysate; lane 4: precipitation of bacteria lysate; ( B ) SDS-PAGE analysis of purified fusion ADAMTS1 and ADAMTS1 on the Coomassie brilliant blue-stained gel; ( C ) Western blot analysis of purified fusion ADAMTS1 and ADAMTS1. Lane 1: purified fusion protein with NTA column; lane 2: the final purified protein after removal of thioredoxin using heparin-sepharose column.

    Article Snippet: Materials E . coli BL21 (DE3), E. coli DH5a, pET32a(+) vector and his-bind purification kit were purchased from Novagen.

    Techniques: Expressing, Recombinant, SDS Page, Purification, Staining, Western Blot

    mFIZZ1 soluble expressed using wheat germ extract. ( A ) The expression of mFIZZ1 in SHuffle™ T7, Origami DE3 and BL21 DE3 analysed on non-reducing 15% SDS-PAGE stained with Coomassie Brilliant Blue. ( B ) Strip of the immunoblot from the SDS-PAGE in ( A ) developed with anti-His antibody is shown. ( C ) The expression of mFIZZ1′ with wheat germ extract analysed on non-reducing 15% SDS-PAGE stained with Coomassie Brilliant Blue. ( D ) Strip of the respective immunoblot of ( C ) developed with anti-His antibody is shown. ( E ) The expression of mFIZZ1 with wheat germ extract analysed on non-reducing 15% SDS-PAGE stained with Coomassie Brilliant Blue. ( F ) Strip of the respective immunoblot of ( E ) developed with anti-His antibody is shown. As marker (M) the PageRuler™ pre-stained Protein Ladder (Fermentas) is used. P = pellet, S = soluble fraction and T = total. The corresponding bands are indicated with an asterisk.

    Journal: PLoS ONE

    Article Title: The Quiescin Sulfhydryl Oxidase (hQSOX1b) Tunes the Expression of Resistin-Like Molecule Alpha (RELM-? or mFIZZ1) in a Wheat Germ Cell-Free Extract

    doi: 10.1371/journal.pone.0055621

    Figure Lengend Snippet: mFIZZ1 soluble expressed using wheat germ extract. ( A ) The expression of mFIZZ1 in SHuffle™ T7, Origami DE3 and BL21 DE3 analysed on non-reducing 15% SDS-PAGE stained with Coomassie Brilliant Blue. ( B ) Strip of the immunoblot from the SDS-PAGE in ( A ) developed with anti-His antibody is shown. ( C ) The expression of mFIZZ1′ with wheat germ extract analysed on non-reducing 15% SDS-PAGE stained with Coomassie Brilliant Blue. ( D ) Strip of the respective immunoblot of ( C ) developed with anti-His antibody is shown. ( E ) The expression of mFIZZ1 with wheat germ extract analysed on non-reducing 15% SDS-PAGE stained with Coomassie Brilliant Blue. ( F ) Strip of the respective immunoblot of ( E ) developed with anti-His antibody is shown. As marker (M) the PageRuler™ pre-stained Protein Ladder (Fermentas) is used. P = pellet, S = soluble fraction and T = total. The corresponding bands are indicated with an asterisk.

    Article Snippet: Plasmid DNA of mFIZZ1 was transformed into E. coli SHuffle™ T7 Express (BioLabs), Origami™ DE3 and BL21 DE3 (Novagen), grown in LB medium supplemented with 25 µg/ml ampicillin, induced at a cell density (OD600 nm ) of 0.7 with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), and cultured for 6 h at 37°C except for SHuffle™ T7 cells, the expression done at 30°C.

    Techniques: Expressing, SDS Page, Staining, Stripping Membranes, Marker

    12% (w/v) SDS-PAGE analysis of recombinant Ldc1E. Lane 1, molecular mass standards. Lane 2, total protein of E . coli BL21 (DE3) pLysS harboring empty pET30a(+) (control). Lane 3, total protein of E . coli BL21 (DE3) pLysS harboring the recombinant ldc1E in pET30a(+). and Lane 4, protein was purified using the Ni-NTA column method. The black arrow indicates the recombinant Ldc1E.

    Journal: PLoS ONE

    Article Title: Identification and molecular characterization of a metagenome-derived L-lysine decarboxylase gene from subtropical soil microorganisms

    doi: 10.1371/journal.pone.0185060

    Figure Lengend Snippet: 12% (w/v) SDS-PAGE analysis of recombinant Ldc1E. Lane 1, molecular mass standards. Lane 2, total protein of E . coli BL21 (DE3) pLysS harboring empty pET30a(+) (control). Lane 3, total protein of E . coli BL21 (DE3) pLysS harboring the recombinant ldc1E in pET30a(+). and Lane 4, protein was purified using the Ni-NTA column method. The black arrow indicates the recombinant Ldc1E.

    Article Snippet: Plasmid pET-30a(+) (Novagen) and bacterial strain E . coli BL21 (DE3) pLysS (Novagen) were used as the expression vector and host, respectively.

    Techniques: SDS Page, Recombinant, Purification

    A fly SUMO-ome: characterization and validation. A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His) 6 -FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The Q SUMO vector, which encodes the mature form of SUMO (SUMO GG ) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, Q ΔGG , which expresses a conjugation defective form of SUMO (SUMO ΔGG ), was used in place of Q SUMO . C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with Q SUMO or Q ΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His) 6 - tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).

    Journal: PLoS ONE

    Article Title: Genetic and Proteomic Evidence for Roles of Drosophila SUMO in Cell Cycle Control, Ras Signaling, and Early Pattern Formation

    doi: 10.1371/journal.pone.0005905

    Figure Lengend Snippet: A fly SUMO-ome: characterization and validation. A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His) 6 -FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The Q SUMO vector, which encodes the mature form of SUMO (SUMO GG ) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, Q ΔGG , which expresses a conjugation defective form of SUMO (SUMO ΔGG ), was used in place of Q SUMO . C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with Q SUMO or Q ΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His) 6 - tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).

    Article Snippet: The bacterial sumoylation assay A vector encoding a candidate SUMO conjugation target fused to GST was co-transformed into BL21 cells (Novagen) with either the QSUMO or the control QΔGG expression vectors.

    Techniques: Isolation, Affinity Purification, Transgenic Assay, Expressing, Purification, Chromatography, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Negative Control, Conjugation Assay, Transformation Assay, Stable Transfection, Western Blot, Lysis, Quantitation Assay

    RP-HPLC separation of the heated cellular extract. Cellular extract of E. coli BL21 [DE3] (pMA24) were treated as described in Section 2.4 . Chromatogram was monitored at (A) λ = 220 nm and (B) λ = 280 nm. Peaks were collected and individually analyzed by MALDI-TOF MS.

    Journal: FEBS Open Bio

    Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria

    doi: 10.1016/j.fob.2012.01.002

    Figure Lengend Snippet: RP-HPLC separation of the heated cellular extract. Cellular extract of E. coli BL21 [DE3] (pMA24) were treated as described in Section 2.4 . Chromatogram was monitored at (A) λ = 220 nm and (B) λ = 280 nm. Peaks were collected and individually analyzed by MALDI-TOF MS.

    Article Snippet: 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C.

    Techniques: High Performance Liquid Chromatography, Mass Spectrometry

    Cascade-mediated interference activity with extended and shortened crRNAs. Shown is the efficiency of plaquing of Type I-Fv CRISPR-Cas system variants expressed in E. coli . The 32 nt spacer sequence (0) was modulated by removal or addition of the indicated number of nucleotides (−18 to +18) while maintaining complementarity to the lambda phage sequence. These arrays were co-expressed in E. coli BL21-AI with pCas6 (in checkers) or pCas7 (Cas3 HD mutant, white bars), which was then subjected to plaque assays with lambda phage. Bars represent mean ± SEM of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system

    doi: 10.1093/nar/gkw469

    Figure Lengend Snippet: Cascade-mediated interference activity with extended and shortened crRNAs. Shown is the efficiency of plaquing of Type I-Fv CRISPR-Cas system variants expressed in E. coli . The 32 nt spacer sequence (0) was modulated by removal or addition of the indicated number of nucleotides (−18 to +18) while maintaining complementarity to the lambda phage sequence. These arrays were co-expressed in E. coli BL21-AI with pCas6 (in checkers) or pCas7 (Cas3 HD mutant, white bars), which was then subjected to plaque assays with lambda phage. Bars represent mean ± SEM of three independent experiments.

    Article Snippet: Briefly, a 1/100 dilution of an E. coli BL21-AI overnight culture was grown until OD600 nm = 0.3 and induced with 0.2% arabinose (Sigma-Aldrich) and 0.1 mM IPTG for 30 min. Then, cells were pelleted and resuspended in 10 mM MgSO4 .

    Techniques: Activity Assay, CRISPR, Sequencing, Mutagenesis

    The S. putrefaciens Type I-Fv CRISPR-Cas system provides heterologous protection against lambda phage infection in E. coli . ( A ) Plaque formation by lambda phage was observed in E. coli BL21-AI strains carrying two plasmids: a first plasmid encoding a crRNA with a spacer of 32 nt complementarity to the lambda phage genome flanked either by a ‘GG’ or ‘GA’ PAM (targeting crRNA) and a second plasmid encoding all Cas proteins (pCas6) or pCas7 (Cas3 HD mutant). ( B ) Quantification of plaque formation was performed in triplicate (represented as efficiency of plaquing, EOP) of strains carrying the WT or the Cas3 HD mutant plasmid, in addition to a second plasmid producing either the targeting crRNA (in the presence of a target ‘GG’ PAM or a target ‘GA’ PAM) or a crRNA with a 32 nt non-targeting random spacer without complementarity to the phage genome. EOP is defined as the ratio between the plaque count of the strain of interest and the strain carrying empty plasmids. Bars represent mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system

    doi: 10.1093/nar/gkw469

    Figure Lengend Snippet: The S. putrefaciens Type I-Fv CRISPR-Cas system provides heterologous protection against lambda phage infection in E. coli . ( A ) Plaque formation by lambda phage was observed in E. coli BL21-AI strains carrying two plasmids: a first plasmid encoding a crRNA with a spacer of 32 nt complementarity to the lambda phage genome flanked either by a ‘GG’ or ‘GA’ PAM (targeting crRNA) and a second plasmid encoding all Cas proteins (pCas6) or pCas7 (Cas3 HD mutant). ( B ) Quantification of plaque formation was performed in triplicate (represented as efficiency of plaquing, EOP) of strains carrying the WT or the Cas3 HD mutant plasmid, in addition to a second plasmid producing either the targeting crRNA (in the presence of a target ‘GG’ PAM or a target ‘GA’ PAM) or a crRNA with a 32 nt non-targeting random spacer without complementarity to the phage genome. EOP is defined as the ratio between the plaque count of the strain of interest and the strain carrying empty plasmids. Bars represent mean ± SEM.

    Article Snippet: Briefly, a 1/100 dilution of an E. coli BL21-AI overnight culture was grown until OD600 nm = 0.3 and induced with 0.2% arabinose (Sigma-Aldrich) and 0.1 mM IPTG for 30 min. Then, cells were pelleted and resuspended in 10 mM MgSO4 .

    Techniques: CRISPR, Infection, Plasmid Preparation, Mutagenesis

    Expression of UspF protein by an IPTG‐inducible E. coli BL21 (DE3) pLyS strain and purification of soluble fractions containing UspF protein. A – T0 total protein extract before induction; T2, total protein extract after induction (3 h); T3, total protein extract after induction (16–18 h). The arrow indicates position of the UspF protein (18.4 kDa). B – Purification of soluble fractions or UspF protein by affinity chromatography using a nickel‐containing resin. 1–Flow through; 2–30 m M , 3–50 m M , 4–100 m M ; 5–200 m M of imidazole.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae

    doi: 10.1002/pro.3038

    Figure Lengend Snippet: Expression of UspF protein by an IPTG‐inducible E. coli BL21 (DE3) pLyS strain and purification of soluble fractions containing UspF protein. A – T0 total protein extract before induction; T2, total protein extract after induction (3 h); T3, total protein extract after induction (16–18 h). The arrow indicates position of the UspF protein (18.4 kDa). B – Purification of soluble fractions or UspF protein by affinity chromatography using a nickel‐containing resin. 1–Flow through; 2–30 m M , 3–50 m M , 4–100 m M ; 5–200 m M of imidazole.

    Article Snippet: Bacterial strains and plasmids The following E. coli K12 strains were used: DH10b (Stratagene, USA) and BL21 (DE3) pLyS (Novagen, USA).

    Techniques: Expressing, Purification, Affinity Chromatography, Flow Cytometry

    SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli BL21 . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively

    Journal: Applied Microbiology and Biotechnology

    Article Title: A novel galactolipase from a green microalga Chlorella kessleri: purification, characterization, molecular cloning, and heterologous expression

    doi: 10.1007/s00253-017-8713-7

    Figure Lengend Snippet: SDS-PAGE analysis of a the purified native ckGL from C. kessleri and b recombinant ckGL (rckGL) produced in E. coli BL21 . Lane M, protein molecular mass markers; lane 1, purified enzyme (2.9 μg protein); lane 2, recombinant enzyme in TF-ckGL form (0.1 μg); lane 3, rckGL (0.1 μg) produced from TF-ckGL by digestion with Factor Xa. The SDS-PAGE gels a and b were stained with Coomassie Brilliant Blue R-250 and Simply Blue Safe Stain (G-250), respectively

    Article Snippet: E. coli JM109 (Takara Bio., Shiga, Japan) was commonly used as the host strain for molecular cloning and strain BL21 (Novagen, Madison, WI) was employed for functional expression of ckGL.

    Techniques: SDS Page, Purification, Recombinant, Produced, Staining

    Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, Escherichia coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.

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

    Article Title: Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase

    doi:

    Figure Lengend Snippet: Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, Escherichia coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.

    Article Snippet: A truncated murine nSMase (mnSMase)2-cDNA construct coding for amino acids 310–655 was fused to a C-terminal 6xHis-tag derived from the plasmid vector pcDNA3.1/Myc-His (Invitrogen) and expressed in Escherichia coli [BL21(DE3)pLysS] using the pET-expression system (Novagen).

    Techniques: Sequencing, Labeling, Binding Assay

    Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, BL21 Gold, BL21 Codon (+) and BL21 Rosetta in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.

    Journal: Protein expression and purification

    Article Title: A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrisanthemi ligand-gated ion channel

    doi: 10.1016/j.pep.2017.03.006

    Figure Lengend Snippet: Optimization of conditions for the overexpression of ELIC 1a . A cartoon representation of ELIC highlighting the membrane spanning pore forming domain (red helices), the extracellular domain (yellow B-sheets), and the boundaries of the cell membrane in relation to the channel structure. 1b . Upper panel, a western blot analysis of the expression levels of the ELIC channel made in the E. coli strains: C41, C43, BL21 Gold, BL21 Codon (+) and BL21 Rosetta in the following growth media: Terrific Broth (TB, yellow), Auto-Induction medium (AI, blue) and LB medium (red). Lower panel, normalized ELIC band density from the western blot (Penta-histidine antibody) were plotted against E. coli strains grouped by growth media. 1c. The effect of the cell culture volume on ELIC expression levels and final culture biomass was studied for the three most promising E. coli strains: BL21 Gold, BL21 Codon (+) and BL21 Rosetta. 1d. The effect of Zn 2+ and Mg 2+ as channel negative modifiers or the competitive antagonist ACh on ELIC expression levels is represented as a bar plot.

    Article Snippet: E. coli strains: BL21 DE3 Gold, BL21 DE3 Codon (+), BL21 DE3 Rosetta (Novagen), C41, and C43 (Lucigen) were tested for their ability to express the MBP-ELIC fusion protein in sufficient quantity and quality.

    Techniques: Over Expression, Western Blot, Expressing, Cell Culture

    Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.

    Journal: BMC Molecular Biology

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

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.

    Article Snippet: Protein expression was performed in E. coli BL21 (DE3) or BL21 (DE3) pLysS Rosetta (Novagen); inducing expression by the addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG, USB) to between 0.1 to 0.5 mM; incubating cultures post-induction at 20-37°C.

    Techniques: Purification, Size-exclusion Chromatography, Plasmid Preparation, SDS Page