bl21 star  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bl21 star
    Cell density at induction and harvesting. Expression tests of archaeal transporters were performed in E. coli C43(DE3) and <t>BL21</t> Star strains. Cells were grown in LB medium, induced with 0.5 mM IPTG at OD 600nm of 0.4-0.8 and harvested 3 hours post-induction. The bars show the OD 600nm at the time of induction (white) and harvesting (black).
    Bl21 Star, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 star/product/Thermo Fisher
    Average 99 stars, based on 46 article reviews
    Price from $9.99 to $1999.99
    bl21 star - by Bioz Stars, 2020-02
    99/100 stars

    Images

    1) Product Images from "An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli"

    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076913

    Cell density at induction and harvesting. Expression tests of archaeal transporters were performed in E. coli C43(DE3) and BL21 Star strains. Cells were grown in LB medium, induced with 0.5 mM IPTG at OD 600nm of 0.4-0.8 and harvested 3 hours post-induction. The bars show the OD 600nm at the time of induction (white) and harvesting (black).
    Figure Legend Snippet: Cell density at induction and harvesting. Expression tests of archaeal transporters were performed in E. coli C43(DE3) and BL21 Star strains. Cells were grown in LB medium, induced with 0.5 mM IPTG at OD 600nm of 0.4-0.8 and harvested 3 hours post-induction. The bars show the OD 600nm at the time of induction (white) and harvesting (black).

    Techniques Used: Expressing

    Affinity purification of seven membrane transport proteins. ( A ) Protein purification by Ni-NTA chromatography of 13 constructs expressed in E. coli BL21 Star. SDS-PAGE (top panel) and Western blot (bottom panel) of purified protein. MW - Bio-rad precision plus protein standards. ( B ) Estimated protein yield per litre of cell culture after initial Ni-NTA affinity chromatography. N/D - not determined.
    Figure Legend Snippet: Affinity purification of seven membrane transport proteins. ( A ) Protein purification by Ni-NTA chromatography of 13 constructs expressed in E. coli BL21 Star. SDS-PAGE (top panel) and Western blot (bottom panel) of purified protein. MW - Bio-rad precision plus protein standards. ( B ) Estimated protein yield per litre of cell culture after initial Ni-NTA affinity chromatography. N/D - not determined.

    Techniques Used: Affinity Purification, Protein Purification, Chromatography, Construct, SDS Page, Western Blot, Purification, Cell Culture, Affinity Chromatography

    Western blot analysis of archaeal membrane transport proteins in E. coli membranes. Membrane transport protein genes cloned into pTTQ18, pET52b and pWarf were tested in two E. coli host strains, C43(DE3) and BL21 Star. Membranes were analysed Western blotting. C - C43(DE3), S - BL21 Star, 1 - pTTQ18, 2 - pET52b(+), 3 - pWarf(-).
    Figure Legend Snippet: Western blot analysis of archaeal membrane transport proteins in E. coli membranes. Membrane transport protein genes cloned into pTTQ18, pET52b and pWarf were tested in two E. coli host strains, C43(DE3) and BL21 Star. Membranes were analysed Western blotting. C - C43(DE3), S - BL21 Star, 1 - pTTQ18, 2 - pET52b(+), 3 - pWarf(-).

    Techniques Used: Western Blot, Clone Assay

    SDS-PAGE analysis of archaeal membrane transport proteins in E. coli membranes. Membrane transport protein genes cloned into pTTQ18, pET52b and pWarf were tested in two E. coli host strains, C43(DE3) and BL21 Star. Membranes were analysed by SDS-PAGE. C - C43(DE3), S - BL21 Star, 1 - pTTQ18, 2 - pET52b(+), 3 - pWarf(-). Positions of the His-tagged protein determined by Western blotting (see Figure 3 ) are indicated by: triangle - protein detected by Western blotting but not visible on SDS-PAGE; star - protein detected by Western blotting and visible on SDS-PAGE gels.
    Figure Legend Snippet: SDS-PAGE analysis of archaeal membrane transport proteins in E. coli membranes. Membrane transport protein genes cloned into pTTQ18, pET52b and pWarf were tested in two E. coli host strains, C43(DE3) and BL21 Star. Membranes were analysed by SDS-PAGE. C - C43(DE3), S - BL21 Star, 1 - pTTQ18, 2 - pET52b(+), 3 - pWarf(-). Positions of the His-tagged protein determined by Western blotting (see Figure 3 ) are indicated by: triangle - protein detected by Western blotting but not visible on SDS-PAGE; star - protein detected by Western blotting and visible on SDS-PAGE gels.

    Techniques Used: SDS Page, Clone Assay, Western Blot

    2) Product Images from "Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins"

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

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

    doi: 10.1073/pnas.0307329101

    Expression of PgbA and PgbB by H. pylori strains of diverse geographical origins. Shown is a Western blot of H. pylori whole cell lysates using Ab 114, which recognizes both PgbA and -B. Lanes: 1, Poland 43; 2, Turkey 33; 3, Iran 23; 4, Sweden 5; 5, Somalia 3; 6, CCUG 17874; 7, 26695; 8, AH244; 9, E. coli BL21 Star. Molecular mass markers (kDa) are indicated.
    Figure Legend Snippet: Expression of PgbA and PgbB by H. pylori strains of diverse geographical origins. Shown is a Western blot of H. pylori whole cell lysates using Ab 114, which recognizes both PgbA and -B. Lanes: 1, Poland 43; 2, Turkey 33; 3, Iran 23; 4, Sweden 5; 5, Somalia 3; 6, CCUG 17874; 7, 26695; 8, AH244; 9, E. coli BL21 Star. Molecular mass markers (kDa) are indicated.

    Techniques Used: Expressing, Western Blot

    3) Product Images from "Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH"

    Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-11-7

    Bioreductions of D-xylose catalyzed by whole cells and crude cell extracts of E. coli strains BL21_XR_FDH and Rosetta_XR_2FDH . Type of catalyst is indicated by symbols: ● Rosetta_XR_2FDH crude extract; ○ Rosetta_XR_2FDH whole-cell catalyst; ∇ BL21_XR_FDH crude extract; ▼ BL21_XR_FDH whole-cell catalyst. Reaction conditions: D-xylose (250 mM), sodium formate (300 mM), cells (10 g CDW /L), 30°C.
    Figure Legend Snippet: Bioreductions of D-xylose catalyzed by whole cells and crude cell extracts of E. coli strains BL21_XR_FDH and Rosetta_XR_2FDH . Type of catalyst is indicated by symbols: ● Rosetta_XR_2FDH crude extract; ○ Rosetta_XR_2FDH whole-cell catalyst; ∇ BL21_XR_FDH crude extract; ▼ BL21_XR_FDH whole-cell catalyst. Reaction conditions: D-xylose (250 mM), sodium formate (300 mM), cells (10 g CDW /L), 30°C.

    Techniques Used:

    4) Product Images from "C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development"

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development

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

    doi: 10.1073/pnas.112276199

    AtCPL are phosphatases. AtCPL1 and AtCPL3 cDNAs were placed under the control of the T7 promoter in the vectors pCPL1 and pCPL3 and expressed in E. coli BL21SI and BL21 star cells to produce rCPL1 and rCPL3, respectively. Protein was isolated from cells without and with pCPL1 or pCPL3 plasmid, normalized based on the culture volume (0.25 ml culture per lane), and then fractionated by SDS/PAGE (6.25% gel). Phosphatase activity was visualized by CCD imaging, after impregnating the gel with CDP star substrate (Zymogram). Protein bands were visualized by Coomassie brilliant blue R-25 (CBB) staining after image acquisition. Innate phosphatase activity of bacterial cells is indicated by *. Zymogram indicates the presence of low M r degradation product of rCPL3 in bacterial cells.
    Figure Legend Snippet: AtCPL are phosphatases. AtCPL1 and AtCPL3 cDNAs were placed under the control of the T7 promoter in the vectors pCPL1 and pCPL3 and expressed in E. coli BL21SI and BL21 star cells to produce rCPL1 and rCPL3, respectively. Protein was isolated from cells without and with pCPL1 or pCPL3 plasmid, normalized based on the culture volume (0.25 ml culture per lane), and then fractionated by SDS/PAGE (6.25% gel). Phosphatase activity was visualized by CCD imaging, after impregnating the gel with CDP star substrate (Zymogram). Protein bands were visualized by Coomassie brilliant blue R-25 (CBB) staining after image acquisition. Innate phosphatase activity of bacterial cells is indicated by *. Zymogram indicates the presence of low M r degradation product of rCPL3 in bacterial cells.

    Techniques Used: Isolation, Plasmid Preparation, SDS Page, Activity Assay, Imaging, Staining

    Related Articles

    Clone Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: For the generation of full-length and deletion constructs of MLP, the PCR products were digested with EcoRI/SalI (Takara Bio, Otsu, Shiga, Japan) restriction enzymes and cloned into the pMAL-c2X vector (New England Biolabs, Beverly, MA). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

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    Article Snippet: To purify the kinase domain of SrrB, primers srrBfwd-NdeI and srrBrev-XhoI were used to amplify the sequence from S. aureus LAC genomic DNA that was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA). .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: The PCR products were cloned into pTrcHis2 as described above. .. Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Centrifugation:

    Article Title: Bioartificial matrices for therapeutic vascularization
    Article Snippet: BL21 Star(DE3) Escherichia coli (Invitrogen) were transformed with VEGF121 -cys plasmid grown in DH5α E. coli (Invitrogen) following manufacturer’s instructions. .. Inclusion bodies containing VEGF121 -cys were separated from soluble protein by centrifugation at 15,000 × g and were solubilized for 1 h on ice in 10 mL B-PER reagent + 6 M urea.

    Amplification:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: Recombinant proteins To generate recombinant His6 -EhDrpA-HA and His6 -EhDrpB-HA proteins, we amplified genes using appropriate primers sets (Supplementary Table ) and pEhEx/EhDrpA-HA and pEhEx/EhDrpB-HA as templates. .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
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    Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH
    Article Snippet: Strains and plasmids The E. coli strains used were JM109 from Promega (Madison, WI, USA), BL21 (DE3) and Rosetta 2 (DE3) from Novagen (VWR International GmbH, Vienna, Austria) and BL21 star (DE3) from Invitrogen (Carlsbad, California, USA). .. The Cb FDH gene was amplified from pETDuet_XR_FDH by a PCR using Pfu DNA polymerase and primers providing Pag I (compatible ends to Nco I) and Avr II restriction sites.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: .. cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: The 9X gene, the wild-type gene, and all nine single-site mutant genes were PCR amplified using primers designed to append an N-terminal hexahistidine tag. .. Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Phosphatase Assay:

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: Paragraph title: Phosphatase Assay. ... cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen).

    Synthesized:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Oligonucleotides (79–80 bases) were synthesized by solid phase oligonucleotide synthesis (MerMade 192; BioAutomation) and assembled into full-length open reading frames (ORFs) by automation , which were reamplified by PCR using 5′-biotinylated primers. .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C.

    Construct:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli
    Article Snippet: .. Over-production of archaeal membrane transport proteins Expression constructs were used to transform two E. coli strains, BL21 Star (Invitrogen) and C43(DE3) [ ], containing the pRARE2 plasmid. .. Expression trials were performed as follows.

    Incubation:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. After eletrophoresis, the gel was incubated in 2.5% Triton X-100 for 30 min and then phosphatase assay buffer (50 mM Tris⋅HCl, pH 7.9/10% glycerol/10 mM MgCl2 /10 mM potassium acetate/1 mM ZnCl2 ) for 30 min.

    Expressing:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: .. A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M). ..

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins
    Article Snippet: .. TOP10 (Invitrogen) and BL21 Star (Invitrogen) were used for protein expression. ..

    Article Title: NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain
    Article Snippet: Paragraph title: Protein Expression and Purification ... Transformed BL21 Star (DE3) One Shot cells (Invitrogen, Barcelona, Spain) were grown in LB medium supplemented with ampicillin at 37 °C.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli
    Article Snippet: .. Over-production of archaeal membrane transport proteins Expression constructs were used to transform two E. coli strains, BL21 Star (Invitrogen) and C43(DE3) [ ], containing the pRARE2 plasmid. .. Expression trials were performed as follows.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins. ... The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Western Blot:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system
    Article Snippet: Paragraph title: GST Pull-Down Assay, Western Blot, and Coimmunoprecipitation. ... GST fusion proteins were purified from BL21 Star (Invitrogen).

    Transformation Assay:

    Article Title: NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain
    Article Snippet: .. Transformed BL21 Star (DE3) One Shot cells (Invitrogen, Barcelona, Spain) were grown in LB medium supplemented with ampicillin at 37 °C. .. Induction of protein expression was initiated when optical density at 600 nm reached 0.6–0.8 by adding 0.4 mM IPTG and zinc chloride 0.1 mM, and progressed for 3–5 h at 23 °C.

    Article Title: Bioartificial matrices for therapeutic vascularization
    Article Snippet: .. BL21 Star(DE3) Escherichia coli (Invitrogen) were transformed with VEGF121 -cys plasmid grown in DH5α E. coli (Invitrogen) following manufacturer’s instructions. .. Transformed BL21 Star(DE3) cells were induced with 1 mM IPTG at OD600 = 0.8.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: .. cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production. ..

    Over Expression:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: Paragraph title: Overexpression and purification of WXG100 proteins as heterodimers. ... A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M).

    Activated Clotting Time Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    High Performance Liquid Chromatography:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

    Protease Inhibitor:

    Article Title: The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system
    Article Snippet: GST fusion proteins were purified from BL21 Star (Invitrogen). .. For Western blot analysis cellular pellets were lysed in ice-cold RIPA buffer (50 mM Tris, pH 7.5/150 mM NaCl/1% Nonidet P-40/0.5% sodium deoxycholate/0.1% SDS) containing Complete Mini protease inhibitor pellet (Roche) and 1 mM PMSF.

    DNA Sequencing:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. All plasmid sequences were verified by bi-directional DNA sequencing.

    Polymerase Chain Reaction:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: For the generation of the CFL2 constructs, PCR products were digested with EcoRI/XhoI (Takara Bio, Otsu, Shiga, Japan) and subcloned into the pGEX-5X vector (Amersham Biosciences Europe). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH
    Article Snippet: Strains and plasmids The E. coli strains used were JM109 from Promega (Madison, WI, USA), BL21 (DE3) and Rosetta 2 (DE3) from Novagen (VWR International GmbH, Vienna, Austria) and BL21 star (DE3) from Invitrogen (Carlsbad, California, USA). .. The Cb FDH gene was amplified from pETDuet_XR_FDH by a PCR using Pfu DNA polymerase and primers providing Pag I (compatible ends to Nco I) and Avr II restriction sites.

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: The PCR products were cloned into pTrcHis2 as described above. .. Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Sonication:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M). .. Cell pellets were resuspended in 15 ml of a buffer containing urea (8 M), Tris-HCl (0.01 M), and NaH2 PO4 (0.1 M) and were lysed by sonication with 10 to 15 bursts (10 s) at maximum power.

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins
    Article Snippet: TOP10 (Invitrogen) and BL21 Star (Invitrogen) were used for protein expression. .. H. pylori CCUG 17874 DNA was sonicated (10 sec on, then 10 sec off over 3–5 min) at maximum power.

    Article Title: NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain
    Article Snippet: Transformed BL21 Star (DE3) One Shot cells (Invitrogen, Barcelona, Spain) were grown in LB medium supplemented with ampicillin at 37 °C. .. Then cells were collected again, and resuspended in Buffer A (Tris HCl 10 mM, DTT 5 mM, glycerol 10%, zinc chloride 100 μM) and broken by sonication.

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Affinity Purification:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Binding Assay:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Immunizations to induce CD4+ T-cell responses were performed with 20-mers containing the core 11 amino acids because MHC class II molecules have open-ended binding pockets and additional amino acids on either side enhances immunoreactivity . .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The following primer sets were used for amplification: 5′-ATC GGA ATT CGT CTT CAA GAT GCC AAA C-3′ and 5′-ATC GGT CGA CTC TGT GCA GGA TTA CTT G-3′ for maltose binding protein (MBP)-MLP (amino acids [aa] 1 to 195), 5′-ATG CGA ATT CAA ATG TGG AGC CTG TGA A-3′and 5′-ACT GGT CGA CGA CTG TTG GAA CTG CAG G-3′ for MBP-MLP-LIM1 (aa 9 to 94), 5′-ACG TGA ATT CAG CAA CCC TTC CAA ATT C-3′ and 5′-ACG TGT CGA CTG TTG TGT AAG GCC TCC A-3′ for MBP-MLP-LIM2 (aa 105 to 188), and 5′-ATG CGA ATT CCG CAG ATA TGG CCC CAA A-3′and 5′-ATC GGT CGA CGG ACT CTC CAA CTT CGC-3′ for MBP-MLP-inter-LIM (aa 64 to 117). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Cellular Antioxidant Activity Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Countercurrent Chromatography:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Pull Down Assay:

    Article Title: The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system
    Article Snippet: Paragraph title: GST Pull-Down Assay, Western Blot, and Coimmunoprecipitation. ... GST fusion proteins were purified from BL21 Star (Invitrogen).

    Mutagenesis:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: Paragraph title: Construction and purification of wild-type and mutant enzymes ... Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Size-exclusion Chromatography:

    Article Title: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins
    Article Snippet: TOP10 (Invitrogen) and BL21 Star (Invitrogen) were used for protein expression. .. H. pylori CCUG 17874 DNA was sonicated (10 sec on, then 10 sec off over 3–5 min) at maximum power.

    Purification:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: Paragraph title: Overexpression and purification of WXG100 proteins as heterodimers. ... A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M).

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain
    Article Snippet: Paragraph title: Protein Expression and Purification ... Transformed BL21 Star (DE3) One Shot cells (Invitrogen, Barcelona, Spain) were grown in LB medium supplemented with ampicillin at 37 °C.

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains. .. The recombinant proteins were purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham Biosciences) for GST fusion proteins or amylose resin beads for MBP fusion proteins (New England Biolabs), according to the manufacturer's instructions.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. Proteins were purified at 4°C from TnT reactions using anti-Flag M2 agarose (Sigma) and Cl− -free buffer: four (15 min) washes of agarose-bound FPs with 20 m m HEPES, pH 7.1, removed Cl− before elution with 120 μl of 3×Flag (Sigma).

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins. ... The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Article Title: The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system
    Article Snippet: .. GST fusion proteins were purified from BL21 Star (Invitrogen). .. GST pull-down assay was performed as described ( , ).

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: Paragraph title: Construction and purification of wild-type and mutant enzymes ... Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask.

    Sequencing:

    Article Title: NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain
    Article Snippet: The 180-residue protease sequence contains a solubilizing C -terminal tail (-SKKKK). .. Transformed BL21 Star (DE3) One Shot cells (Invitrogen, Barcelona, Spain) were grown in LB medium supplemented with ampicillin at 37 °C.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: .. cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: To purify the kinase domain of SrrB, primers srrBfwd-NdeI and srrBrev-XhoI were used to amplify the sequence from S. aureus LAC genomic DNA that was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA). .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Protein Extraction:

    Article Title: Bioartificial matrices for therapeutic vascularization
    Article Snippet: BL21 Star(DE3) Escherichia coli (Invitrogen) were transformed with VEGF121 -cys plasmid grown in DH5α E. coli (Invitrogen) following manufacturer’s instructions. .. Pelleted cells were thawed and lysed in bacterial protein extraction reagent (B-PER) protein extraction reagent (Pierce).

    Positron Emission Tomography:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Recombinant:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: Paragraph title: Construction of recombinant proteins. ... Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG. .. After induction, target proteins were purified from bacterial lysates by Ni-NTA system (QIAGEN GmbH, Hilden, Germany).

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Expression and purification of recombinant proteins. ... The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production.

    Staining:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. Protein expression and purity relative to a sample of the Clomeleon chloride-sensitive YFP Topaz domain (CT) was determined by densitometry of the YFP band in protein gels stained with GelCode Blue (Thermo Scientific) using ImageJ (release 1.42q) software (good category of expression > 90% pure; poorly expressed proteins were less pure).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The primers for CFL2 amplification were as follows: 5′-ATG CGA ATT CAT GGC TTC TGG AGT TAC A-3′and 5′-ATG CCT CGA GTG GCA CTT GAC TGT CAT T-3′ for GST-CFL2 (aa 1 to 166), 5′-CCG TGA ATT CAC ATG GCT TCT GGA GTT-3′ and 5′-ACG TCT CGA GCA AGA TCT GCT TTG CTT C-3′ for GST-CFL2-A (aa 1 to 55), 5′-ACG TGA ATT CAA GTC ATC AAA GTT T-3′and 5′-ACG TCT CGA GCT ACT ACA TTG CCT C-3′ for GST-CFL2-B (aa 105 to 166), and 5′-ATG CGA ATT CAG ATC TTG GTG GGT GAC-3′ and 5′-ATG CCT CGA GAC TTT CAG GAG CCC A-3′ for GST-CFL2-C (aa 55 to 108). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    SDS Page:

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. AtCPL proteins were prepared in nonreducing SDS/PAGE loading buffer and size-fractioned in a 6.25% SDS/PAGE gel ( ).

    Article Title: The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system
    Article Snippet: GST fusion proteins were purified from BL21 Star (Invitrogen). .. Lysates were electrophoresed on SDS/PAGE gels and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech).

    Plasmid Preparation:

    Article Title: Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA ▿ †
    Article Snippet: .. A 5-ml starter culture of BL21 Star (Invitrogen) cells carrying the expression plasmid was inoculated into 400 ml of broth, grown at 37°C to an optical density at 600 nm of 0.7, and induced with isopropyl-β- d -thiogalactopyranoside (IPTG) (3 × 10−4 M). ..

    Article Title: NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain
    Article Snippet: Protein Expression and Purification NS3 protease from HCV genotype 1b J-strain is encoded in a pET7 plasmid (a kind gift from Dr. C. Steinkühler, Istituto di Ricerche Molecolare, P.Angeletti, Rome). .. Transformed BL21 Star (DE3) One Shot cells (Invitrogen, Barcelona, Spain) were grown in LB medium supplemented with ampicillin at 37 °C.

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: For the generation of the CFL2 constructs, PCR products were digested with EcoRI/XhoI (Takara Bio, Otsu, Shiga, Japan) and subcloned into the pGEX-5X vector (Amersham Biosciences Europe). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Article Title: Bioartificial matrices for therapeutic vascularization
    Article Snippet: .. BL21 Star(DE3) Escherichia coli (Invitrogen) were transformed with VEGF121 -cys plasmid grown in DH5α E. coli (Invitrogen) following manufacturer’s instructions. .. Transformed BL21 Star(DE3) cells were induced with 1 mM IPTG at OD600 = 0.8.

    Article Title: Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica
    Article Snippet: Fragments were digested by appropriate sets of restriction enzymes and ligated into pCold™ I plasmid (TaKaRa). .. These plasmids were transformed into BL21 Star™ (DE3) One Shot® Chemically Competent E . coli (Invitrogen) and expression of recombinant proteins was induced by low temperature (15 °C) with 1 mM IPTG.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. Expression and purification of recombinant OGG1 and APE1 proteins The human OGG1 1a coding sequence in pET-28a expression vector (Novagen) was used to express his-tagged OGG1 in BL21 Codon Plus (Stratagene), Rosetta Blue (Novagen) and BL21 Star (Invitrogen). pET-28a plasmids containing the wild-type CΔ19, S326C, S326C CΔ19 and CΔ20 OGG1 coding sequence were created with a Quik Change II XL mutagenesis kit (Stratagene) or PCR. .. Mammalian expression vectors encoding N-terminally FLAG-tagged OGG1s were constructed by cloning PCR amplified OGG1 genes into pCMV-2B expression vector (Stratagene).

    Article Title: An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli
    Article Snippet: .. Over-production of archaeal membrane transport proteins Expression constructs were used to transform two E. coli strains, BL21 Star (Invitrogen) and C43(DE3) [ ], containing the pRARE2 plasmid. .. Expression trials were performed as follows.

    Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH
    Article Snippet: Strains and plasmids The E. coli strains used were JM109 from Promega (Madison, WI, USA), BL21 (DE3) and Rosetta 2 (DE3) from Novagen (VWR International GmbH, Vienna, Austria) and BL21 star (DE3) from Invitrogen (Carlsbad, California, USA). .. The construction of the E. coli BL21 (DE3) (BL21_XR_FDH) harbouring Ct XR and Cb FDH genes on a pETDuet-1 vector (pETDuet_XR_FDH) was described elsewhere [ ].

    Article Title: C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development
    Article Snippet: cDNA fragments encoding AtCPL1 and AtCPL3 were amplified by Pfx polymerase with primers containing attB1 (5′ primers) and attB2 (3′ primers) sequence (B1C15 and B2C13, B1C35 and B2C33, Table 1) and recombined into pDONR201 and then recombined into pDEST17 according to the manufacturer's protocol (Invitrogen) to produce pDCPL1 and pDCPL3. pDCPL1 was transformed into Escherichia coli BL21SI and pDCPL3 into BL21-star (Invitrogen). .. For recombinant protein synthesis, BL21SI cells without and with pDCPL1 plasmid were induced for 10 h by adding 0.3 M NaCl into the medium. pDCPL3 was introduced into BL21 star because of low AtCPL3 expression in BL21SI, and cells were induced with 1 mM isopropyl β- d -thiogalactoside for 10 h. The recombinant AtCPL1 and AtCPL3 proteins were recovered in the insoluble fraction after sonication of bacterial cells in 50 mM Tris (pH 7.5) buffer and extensive washes with 1% Triton X-100.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: .. The pMW17 vector was transformed into BL21 Star(DE3) (Invitrogen, Carlsbad, CA) cells for protein production. ..

    Software:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. Protein expression and purity relative to a sample of the Clomeleon chloride-sensitive YFP Topaz domain (CT) was determined by densitometry of the YFP band in protein gels stained with GelCode Blue (Thermo Scientific) using ImageJ (release 1.42q) software (good category of expression > 90% pure; poorly expressed proteins were less pure).

    Affinity Chromatography:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains. .. The recombinant proteins were purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham Biosciences) for GST fusion proteins or amylose resin beads for MBP fusion proteins (New England Biolabs), according to the manufacturer's instructions.

    In Vitro:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Produced:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific). .. Expression was confirmed by using anti-V5 monoclonal antibodies by western blotting and purified by 6xHis tag elution using nickel resins (Promega).

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C. .. All proteins had combined C-terminal Flag and His6 peptides for affinity purification.

    Oligonucleotide Synthesis:

    Article Title: Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
    Article Snippet: Oligonucleotides (79–80 bases) were synthesized by solid phase oligonucleotide synthesis (MerMade 192; BioAutomation) and assembled into full-length open reading frames (ORFs) by automation , which were reamplified by PCR using 5′-biotinylated primers. .. Protein was produced by in vitro -coupled transcription and translation (TnT) reactions ( ): 2.4 μg of linear, biotinylated dsDNA was added to 30 μl of a BL21 Star (DE3; Invitrogen) cell lysate and a solution of amino acids, nucleotide triphosphates, and cofactors and water (final volume 120 μl) in 96-well PCR plates, which were covered with a breathable seal for optimum aerobic expression and chromophore maturation , and incubated with shaking for 8 h at 30°C, followed by 6 h at 4°C.

    CTG Assay:

    Article Title: Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle ▿
    Article Snippet: The following primer sets were used for amplification: 5′-ATC GGA ATT CGT CTT CAA GAT GCC AAA C-3′ and 5′-ATC GGT CGA CTC TGT GCA GGA TTA CTT G-3′ for maltose binding protein (MBP)-MLP (amino acids [aa] 1 to 195), 5′-ATG CGA ATT CAA ATG TGG AGC CTG TGA A-3′and 5′-ACT GGT CGA CGA CTG TTG GAA CTG CAG G-3′ for MBP-MLP-LIM1 (aa 9 to 94), 5′-ACG TGA ATT CAG CAA CCC TTC CAA ATT C-3′ and 5′-ACG TGT CGA CTG TTG TGT AAG GCC TCC A-3′ for MBP-MLP-LIM2 (aa 105 to 188), and 5′-ATG CGA ATT CCG CAG ATA TGG CCC CAA A-3′and 5′-ATC GGT CGA CGG ACT CTC CAA CTT CGC-3′ for MBP-MLP-inter-LIM (aa 64 to 117). .. Glutathione S -transferase (GST) and MBP fusion proteins were expressed by induction with 0.5 mM isopropyl-β- d -thiogalactopyranoside for 4 h in the BL21 Star (Invitrogen) and library efficient DH5α (Invitrogen) Escherichia coli strains.

    Lysis:

    Article Title: An evolutionary route to xylanase process fitness
    Article Snippet: Overnight cultures of BL21 Star (Invitrogen) cells carrying the appropriate gene in pTrcHis2 were used to inoculate 1.5 L of Terrific broth (12 g/liter Bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 12.5 g/L K2 HPO4 , 2.3 g/L KH2 PO4 ) containing 100 μg/mL ampicillin in a six-liter shake flask. .. The pellet was resuspended in lysis buffer (50 mM potassium phosphate [pH 7.0], 100 mM KCl, 7.5 mM β-mercaptoethanol) and cells were lysed by French press (15,000 psi).

    Variant Assay:

    Article Title: A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity
    Article Snippet: The residue numbers, in subscript, and peptide sequences, in brackets, of the peptides used are: mouse MPO409–428 peptide (PRWNGEKLYQEARKIVGAMV), Treponema vincentii hypothetical protein167–186 (LRKQLKRLYKEARKIQKCIP), Aspergillus fumigatus HEAT repeat protein825–844 (ISALPQRWYQEARKIIFEAA), Helicobacter pylori RNA polymerase factor sigma-54163–182 (RELDNNELYEEARKIILNLE), Bacteroides sp . chloramphenicol O-acetyltransferase104–123 (YHEDFETFYQEARKIIDSIP), S. aureus pSJH101-derived 6PGD391–410 (YFKNIVTDYQEALRDVVATG), S. aureus 6PGD391–410 Variant 1 (YFKNIVTDYQDALRDVVATG), S. aureus 6PGD391–410 Variant 2 (YFKNIVTNYQEALRDVVATG), S. aureus 6PGD391–410 Variant 3 (YFKNIVTEYQDALRDVVATG), S. aureus 6PGD391–410 Variant 4 (YFKNIVTNYQDALRDVVATG), Mus musculus 6PGD394–413 (FFKSAVDNCQDSWRRVISTGV), and control OVA323–339 peptide (ISQAVHAAHAEINEAGR). .. Recombinant S. aureus pSJH101 6PGD (Genbank ID: CP000737.1) (GeneArt®, ThermoFisher Scientific) was produced using the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot chemically competent E. Coli (ThermoFisher Scientific).

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