nico21 de3  (New England Biolabs)


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    New England Biolabs nico21 de3
    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. <t>NiCo21(DE3)</t> E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
    Nico21 De3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli"

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-107

    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. NiCo21(DE3) E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
    Figure Legend Snippet: SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. NiCo21(DE3) E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.

    Techniques Used: SDS Page, Western Blot, Recombinant, Transformation Assay, Plasmid Preparation, Labeling, Staining

    Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. col i cultures (0.6OD 600 ) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A) . Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD 600 ) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B) . Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).
    Figure Legend Snippet: Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. col i cultures (0.6OD 600 ) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A) . Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD 600 ) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B) . Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).

    Techniques Used: Concentration Assay, Expressing, Transformation Assay, Incubation, Western Blot, Staining

    Effect of rare tRNA supplementation on HIV-1 CA expression in NiCo21(DE3) E. coli. Rare codon analysis of open reading frame (ORF) coding for HIV-1 p24 gene (A) . NiCo21(DE3) E. coli were transformed with pACYC-RIL or pRARE2 or pRARE2-lysS and subsequently transformed with pSA-Hp24-6His vectors and selected on LB + Cam + Amp plates. Cultures were grown in presence of Cam + Amp at 22°C for 12 hours, either induced with 0.05 mM IPTG, or un-induced. Cultures were processed to obtain whole cell lysate (WCL), insoluble (S), and soluble (S) fractions. Samples were electrophoresed on 12% gel, stained with Coomassie Blue G250, and photographed (B) . Lane M, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction.
    Figure Legend Snippet: Effect of rare tRNA supplementation on HIV-1 CA expression in NiCo21(DE3) E. coli. Rare codon analysis of open reading frame (ORF) coding for HIV-1 p24 gene (A) . NiCo21(DE3) E. coli were transformed with pACYC-RIL or pRARE2 or pRARE2-lysS and subsequently transformed with pSA-Hp24-6His vectors and selected on LB + Cam + Amp plates. Cultures were grown in presence of Cam + Amp at 22°C for 12 hours, either induced with 0.05 mM IPTG, or un-induced. Cultures were processed to obtain whole cell lysate (WCL), insoluble (S), and soluble (S) fractions. Samples were electrophoresed on 12% gel, stained with Coomassie Blue G250, and photographed (B) . Lane M, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction.

    Techniques Used: Expressing, Transformation Assay, Staining

    Effect of cultivation medium composition on production of HIV-1 CA. NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in various cultivation media supplemented with 1% glucose and 0.05 mM IPTG at 22°C for 12 hours. Cultures were adjusted to 1.5OD 600 (to normalize the biomass) and 8 uL of culture was analyzed using SDS-PAGE (A) . M, Pre-stained protein ladder; Lanes UI, un-induced; Lanes I, IPTG-induced. Comparison of HIV-1 CA production levels obtained using different growth media (B) . Cells were harvested, lysed, and subjected to Chitin/IMAC purification. Eluted protein was quantitated and HIV-1 CA produced from 1L of biomass was calculated.
    Figure Legend Snippet: Effect of cultivation medium composition on production of HIV-1 CA. NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in various cultivation media supplemented with 1% glucose and 0.05 mM IPTG at 22°C for 12 hours. Cultures were adjusted to 1.5OD 600 (to normalize the biomass) and 8 uL of culture was analyzed using SDS-PAGE (A) . M, Pre-stained protein ladder; Lanes UI, un-induced; Lanes I, IPTG-induced. Comparison of HIV-1 CA production levels obtained using different growth media (B) . Cells were harvested, lysed, and subjected to Chitin/IMAC purification. Eluted protein was quantitated and HIV-1 CA produced from 1L of biomass was calculated.

    Techniques Used: Expressing, SDS Page, Staining, Purification, Produced

    Purification of HIV-1 CA expressed in NiCo21(DE3) E. coli . NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in presence of 0.05 mM IPTG at 22°C for 12 hours and processed to purify recombinant protein using Co 2+ resin, chitin resin, and Chitin + Co 2+ resins. Sample collected during the procedure were analyzed by SDS-PAGE (A) and western blot (B) . Lane M, BenchMark Pre-stained protein ladder; Lane M1, MagicMark XP Western Protein Standard; Lane 1, CA purified on Co 2+ resin; Lane 2, CA purified on chitin resin; Lane 3, CA sequentially purified on chitin and Co 2+ resins. Red arrowheads show contaminating proteins when CA was purified using Co 2+ resin alone (Lane 1).
    Figure Legend Snippet: Purification of HIV-1 CA expressed in NiCo21(DE3) E. coli . NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in presence of 0.05 mM IPTG at 22°C for 12 hours and processed to purify recombinant protein using Co 2+ resin, chitin resin, and Chitin + Co 2+ resins. Sample collected during the procedure were analyzed by SDS-PAGE (A) and western blot (B) . Lane M, BenchMark Pre-stained protein ladder; Lane M1, MagicMark XP Western Protein Standard; Lane 1, CA purified on Co 2+ resin; Lane 2, CA purified on chitin resin; Lane 3, CA sequentially purified on chitin and Co 2+ resins. Red arrowheads show contaminating proteins when CA was purified using Co 2+ resin alone (Lane 1).

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

    nico21 de3  (New England Biolabs)


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    Structured Review

    New England Biolabs nico21 de3
    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. <t>NiCo21(DE3)</t> E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
    Nico21 De3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli"

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-107

    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. NiCo21(DE3) E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
    Figure Legend Snippet: SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. NiCo21(DE3) E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.

    Techniques Used: SDS Page, Western Blot, Recombinant, Transformation Assay, Plasmid Preparation, Labeling, Staining

    Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. col i cultures (0.6OD 600 ) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A) . Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD 600 ) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B) . Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).
    Figure Legend Snippet: Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. col i cultures (0.6OD 600 ) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A) . Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD 600 ) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B) . Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).

    Techniques Used: Concentration Assay, Expressing, Transformation Assay, Incubation, Western Blot, Staining

    Effect of rare tRNA supplementation on HIV-1 CA expression in NiCo21(DE3) E. coli. Rare codon analysis of open reading frame (ORF) coding for HIV-1 p24 gene (A) . NiCo21(DE3) E. coli were transformed with pACYC-RIL or pRARE2 or pRARE2-lysS and subsequently transformed with pSA-Hp24-6His vectors and selected on LB + Cam + Amp plates. Cultures were grown in presence of Cam + Amp at 22°C for 12 hours, either induced with 0.05 mM IPTG, or un-induced. Cultures were processed to obtain whole cell lysate (WCL), insoluble (S), and soluble (S) fractions. Samples were electrophoresed on 12% gel, stained with Coomassie Blue G250, and photographed (B) . Lane M, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction.
    Figure Legend Snippet: Effect of rare tRNA supplementation on HIV-1 CA expression in NiCo21(DE3) E. coli. Rare codon analysis of open reading frame (ORF) coding for HIV-1 p24 gene (A) . NiCo21(DE3) E. coli were transformed with pACYC-RIL or pRARE2 or pRARE2-lysS and subsequently transformed with pSA-Hp24-6His vectors and selected on LB + Cam + Amp plates. Cultures were grown in presence of Cam + Amp at 22°C for 12 hours, either induced with 0.05 mM IPTG, or un-induced. Cultures were processed to obtain whole cell lysate (WCL), insoluble (S), and soluble (S) fractions. Samples were electrophoresed on 12% gel, stained with Coomassie Blue G250, and photographed (B) . Lane M, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction.

    Techniques Used: Expressing, Transformation Assay, Staining

    Effect of cultivation medium composition on production of HIV-1 CA. NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in various cultivation media supplemented with 1% glucose and 0.05 mM IPTG at 22°C for 12 hours. Cultures were adjusted to 1.5OD 600 (to normalize the biomass) and 8 uL of culture was analyzed using SDS-PAGE (A) . M, Pre-stained protein ladder; Lanes UI, un-induced; Lanes I, IPTG-induced. Comparison of HIV-1 CA production levels obtained using different growth media (B) . Cells were harvested, lysed, and subjected to Chitin/IMAC purification. Eluted protein was quantitated and HIV-1 CA produced from 1L of biomass was calculated.
    Figure Legend Snippet: Effect of cultivation medium composition on production of HIV-1 CA. NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in various cultivation media supplemented with 1% glucose and 0.05 mM IPTG at 22°C for 12 hours. Cultures were adjusted to 1.5OD 600 (to normalize the biomass) and 8 uL of culture was analyzed using SDS-PAGE (A) . M, Pre-stained protein ladder; Lanes UI, un-induced; Lanes I, IPTG-induced. Comparison of HIV-1 CA production levels obtained using different growth media (B) . Cells were harvested, lysed, and subjected to Chitin/IMAC purification. Eluted protein was quantitated and HIV-1 CA produced from 1L of biomass was calculated.

    Techniques Used: Expressing, SDS Page, Staining, Purification, Produced

    Purification of HIV-1 CA expressed in NiCo21(DE3) E. coli . NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in presence of 0.05 mM IPTG at 22°C for 12 hours and processed to purify recombinant protein using Co 2+ resin, chitin resin, and Chitin + Co 2+ resins. Sample collected during the procedure were analyzed by SDS-PAGE (A) and western blot (B) . Lane M, BenchMark Pre-stained protein ladder; Lane M1, MagicMark XP Western Protein Standard; Lane 1, CA purified on Co 2+ resin; Lane 2, CA purified on chitin resin; Lane 3, CA sequentially purified on chitin and Co 2+ resins. Red arrowheads show contaminating proteins when CA was purified using Co 2+ resin alone (Lane 1).
    Figure Legend Snippet: Purification of HIV-1 CA expressed in NiCo21(DE3) E. coli . NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in presence of 0.05 mM IPTG at 22°C for 12 hours and processed to purify recombinant protein using Co 2+ resin, chitin resin, and Chitin + Co 2+ resins. Sample collected during the procedure were analyzed by SDS-PAGE (A) and western blot (B) . Lane M, BenchMark Pre-stained protein ladder; Lane M1, MagicMark XP Western Protein Standard; Lane 1, CA purified on Co 2+ resin; Lane 2, CA purified on chitin resin; Lane 3, CA sequentially purified on chitin and Co 2+ resins. Red arrowheads show contaminating proteins when CA was purified using Co 2+ resin alone (Lane 1).

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

    escherichia coli nico21 de3 cells  (New England Biolabs)


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    New England Biolabs escherichia coli nico21 de3 cells
    Escherichia Coli Nico21 De3 Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    e coli nico21 de3 strain  (New England Biolabs)


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    New England Biolabs e coli nico21 de3 strain
    E Coli Nico21 De3 Strain, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    competent nico21 de3 cells  (New England Biolabs)


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    New England Biolabs competent nico21 de3 cells
    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
    Competent Nico21 De3 Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae"

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    Journal: Microbial Genomics

    doi: 10.1099/mgen.0.000594

    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
    Figure Legend Snippet: Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.

    Techniques Used: Sequencing, Binding Assay

    nico21 de3  (New England Biolabs)


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    New England Biolabs nico21 de3
    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
    Nico21 De3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    1) Product Images from "gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae"

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    Journal: Microbial Genomics

    doi: 10.1099/mgen.0.000594

    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
    Figure Legend Snippet: Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.

    Techniques Used: Sequencing, Binding Assay

    e coli strain nico21 de3  (New England Biolabs)


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    New England Biolabs nico21 de3
    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. <t>NiCo21(DE3)</t> E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
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    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. <t>NiCo21(DE3)</t> E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
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    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. <t>NiCo21(DE3)</t> E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.
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    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
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    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
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    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
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    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.
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    SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. NiCo21(DE3) E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.

    Journal: BMC Biotechnology

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    doi: 10.1186/1472-6750-13-107

    Figure Lengend Snippet: SDS-PAGE/immunoblot analysis of recombinant HIV-1 CA. NiCo21(DE3) E. coli were transformed with pSA-Hp24-6His vector and grown at 30°C for 6 hours, either induced with 0.4 mM IPTG, or un-induced. Cells were processed as described in Methods and labeled as whole cell lysate (WCL), insoluble fraction (IS), and soluble fraction (S). Eight microliter of each sample (WCL, IS, S) was mixed with 4X loading dye and electrophoresed on 12% gel. Proteins were transferred to nitrocellulose membrane and stained with MemCode™ Reversible Protein Stain Kit (Pierce) and photographed (left panel) . Membrane was then destained and subjected to immunoblot analysis using primary anti-p24 antibody and secondary anti-Mouse antibody. Membrane was developed using enhanced chemilumenscent reagent (Pierce) and image was captured (right panel) . Lane M1, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction; Lane M2, MagicMark XP Western Protein Standard.

    Article Snippet: The E. coli strains DH5α (NEB #C2987H) and NiCo21(DE3) (NEB #C2529H) were used for cloning and expression experiments respectively.

    Techniques: SDS Page, Western Blot, Recombinant, Transformation Assay, Plasmid Preparation, Labeling, Staining

    Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. col i cultures (0.6OD 600 ) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A) . Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD 600 ) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B) . Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).

    Journal: BMC Biotechnology

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    doi: 10.1186/1472-6750-13-107

    Figure Lengend Snippet: Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. col i cultures (0.6OD 600 ) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A) . Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD 600 ) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B) . Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).

    Article Snippet: The E. coli strains DH5α (NEB #C2987H) and NiCo21(DE3) (NEB #C2529H) were used for cloning and expression experiments respectively.

    Techniques: Concentration Assay, Expressing, Transformation Assay, Incubation, Western Blot, Staining

    Effect of rare tRNA supplementation on HIV-1 CA expression in NiCo21(DE3) E. coli. Rare codon analysis of open reading frame (ORF) coding for HIV-1 p24 gene (A) . NiCo21(DE3) E. coli were transformed with pACYC-RIL or pRARE2 or pRARE2-lysS and subsequently transformed with pSA-Hp24-6His vectors and selected on LB + Cam + Amp plates. Cultures were grown in presence of Cam + Amp at 22°C for 12 hours, either induced with 0.05 mM IPTG, or un-induced. Cultures were processed to obtain whole cell lysate (WCL), insoluble (S), and soluble (S) fractions. Samples were electrophoresed on 12% gel, stained with Coomassie Blue G250, and photographed (B) . Lane M, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction.

    Journal: BMC Biotechnology

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    doi: 10.1186/1472-6750-13-107

    Figure Lengend Snippet: Effect of rare tRNA supplementation on HIV-1 CA expression in NiCo21(DE3) E. coli. Rare codon analysis of open reading frame (ORF) coding for HIV-1 p24 gene (A) . NiCo21(DE3) E. coli were transformed with pACYC-RIL or pRARE2 or pRARE2-lysS and subsequently transformed with pSA-Hp24-6His vectors and selected on LB + Cam + Amp plates. Cultures were grown in presence of Cam + Amp at 22°C for 12 hours, either induced with 0.05 mM IPTG, or un-induced. Cultures were processed to obtain whole cell lysate (WCL), insoluble (S), and soluble (S) fractions. Samples were electrophoresed on 12% gel, stained with Coomassie Blue G250, and photographed (B) . Lane M, BenchMark Pre-stained protein ladder; Lanes WCL, whole cell lysate; Lanes IS, insoluble fraction; Lanes S, soluble fraction.

    Article Snippet: The E. coli strains DH5α (NEB #C2987H) and NiCo21(DE3) (NEB #C2529H) were used for cloning and expression experiments respectively.

    Techniques: Expressing, Transformation Assay, Staining

    Effect of cultivation medium composition on production of HIV-1 CA. NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in various cultivation media supplemented with 1% glucose and 0.05 mM IPTG at 22°C for 12 hours. Cultures were adjusted to 1.5OD 600 (to normalize the biomass) and 8 uL of culture was analyzed using SDS-PAGE (A) . M, Pre-stained protein ladder; Lanes UI, un-induced; Lanes I, IPTG-induced. Comparison of HIV-1 CA production levels obtained using different growth media (B) . Cells were harvested, lysed, and subjected to Chitin/IMAC purification. Eluted protein was quantitated and HIV-1 CA produced from 1L of biomass was calculated.

    Journal: BMC Biotechnology

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    doi: 10.1186/1472-6750-13-107

    Figure Lengend Snippet: Effect of cultivation medium composition on production of HIV-1 CA. NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in various cultivation media supplemented with 1% glucose and 0.05 mM IPTG at 22°C for 12 hours. Cultures were adjusted to 1.5OD 600 (to normalize the biomass) and 8 uL of culture was analyzed using SDS-PAGE (A) . M, Pre-stained protein ladder; Lanes UI, un-induced; Lanes I, IPTG-induced. Comparison of HIV-1 CA production levels obtained using different growth media (B) . Cells were harvested, lysed, and subjected to Chitin/IMAC purification. Eluted protein was quantitated and HIV-1 CA produced from 1L of biomass was calculated.

    Article Snippet: The E. coli strains DH5α (NEB #C2987H) and NiCo21(DE3) (NEB #C2529H) were used for cloning and expression experiments respectively.

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

    Purification of HIV-1 CA expressed in NiCo21(DE3) E. coli . NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in presence of 0.05 mM IPTG at 22°C for 12 hours and processed to purify recombinant protein using Co 2+ resin, chitin resin, and Chitin + Co 2+ resins. Sample collected during the procedure were analyzed by SDS-PAGE (A) and western blot (B) . Lane M, BenchMark Pre-stained protein ladder; Lane M1, MagicMark XP Western Protein Standard; Lane 1, CA purified on Co 2+ resin; Lane 2, CA purified on chitin resin; Lane 3, CA sequentially purified on chitin and Co 2+ resins. Red arrowheads show contaminating proteins when CA was purified using Co 2+ resin alone (Lane 1).

    Journal: BMC Biotechnology

    Article Title: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

    doi: 10.1186/1472-6750-13-107

    Figure Lengend Snippet: Purification of HIV-1 CA expressed in NiCo21(DE3) E. coli . NiCo21(DE3)-pACYC-RIL expressing HIV-1 CA were grown in presence of 0.05 mM IPTG at 22°C for 12 hours and processed to purify recombinant protein using Co 2+ resin, chitin resin, and Chitin + Co 2+ resins. Sample collected during the procedure were analyzed by SDS-PAGE (A) and western blot (B) . Lane M, BenchMark Pre-stained protein ladder; Lane M1, MagicMark XP Western Protein Standard; Lane 1, CA purified on Co 2+ resin; Lane 2, CA purified on chitin resin; Lane 3, CA sequentially purified on chitin and Co 2+ resins. Red arrowheads show contaminating proteins when CA was purified using Co 2+ resin alone (Lane 1).

    Article Snippet: The E. coli strains DH5α (NEB #C2987H) and NiCo21(DE3) (NEB #C2529H) were used for cloning and expression experiments respectively.

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

    Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.

    Journal: Microbial Genomics

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    doi: 10.1099/mgen.0.000594

    Figure Lengend Snippet: Strains, plasmids and oligonucleotides used in this study Restriction enzyme recognition sites are underlined. The primer sequence incorporating a C-terminal 6xHis translational fusion into chiA-3 is presented in lowercase, and the sequences of ribosome binding sites, start and STOP codons are in bold. CmR, chloramphenicol resistant; StrR, streptomycin resistant.

    Article Snippet: Sequence-verified plasmids were transformed into chemically competent NiCo21(DE3) cells (NEB; #C2529H) following the manufacturer’s instructions, and these transformants were used for protein expression purposes.

    Techniques: Sequencing, Binding Assay