bl21 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bl21 cells
    Bl21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 cells/product/Thermo Fisher
    Average 90 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    bl21 cells - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s
    Article Snippet: Kinesin proteins expression Kinesin proteins were expressed in BL21 cells (BL21(DE3)pLysS, Thermofisher). .. The neck motor construct (NM) includes KLP10A residues 198–615 cloned into pRSET B plasmid with an N-terminal histidine tag.

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: Production of polyclonal antiserum to gpD The authentic (non-codon-optimized) λ phage gpD gene was amplified by PCR from pAT101 (kindly provided by Dr Andreas Plückthun and Dr Patrick Forrer, University of Zurich, Switzerland) ( , ) using the gpD forward primer (5′-ggtgcc catatg gcgagcaaagaaacctttacc-3′) and the gpD reverse primer (5′-ccgacg ggatcc tcattaaacgatgctgattgc-3′) and then cloned into the pET15b plasmid (Novagen) using the NdeI and BamHI restriction sites (underlined). .. The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen).

    Article Title: When a Module is not a Domain – the Case of the REJ Module and the Redefinition of the Architecture of Polycystin-1
    Article Snippet: Paragraph title: Cloning and protein expression ... For protein expression in inclusion bodies constructs were transformed into BL21* cells (Invitrogen).

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: We designed a DNA fragment encoding the forward linker SGGSTS, the Z-domain of protein A, the backward linker ASTGS, and cysteine, synthesized by Fasmac Co., Ltd. (Kanagawa, Japan), which was then cloned into pGEX-6P-1 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden).

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen). .. Expression was induced with IPTG (isopropyl-β- d -thiogalactopyranoside; 1 mM) for 18 h at 22°C.

    Article Title: The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo
    Article Snippet: Monoclonal antibody generation The protein region of UNC-104 (amino acid 740-1117) was cloned into pRSETA vector (Invitrogen) using standard techniques. .. Protein was expressed in BL21 cells (Invitrogen), and purified using Ni-NTA chromatography (QIAGEN).

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: .. The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen. .. Antiserum against recombinant ggLRP2 was raised in adult female New Zealand White rabbits by injections of antigen, 200 mg each, as described previously ( ).

    Centrifugation:

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen). .. Cells were pelleted by centrifugation, lysed with BugBuster (Novagen), and treated with benzonase and lysozyme (Novagen) according to the manufacturer's recommendations.

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. .. Bacterial cells were pelleted at 6000 rpm for 10 min and lysed in lysis buffer (1 M KCl, 50 mM Tris pH 7.4, 10 mM imidazole, 1 mM CaCl2 , 5% glycerol, 1% NP40, 1.5 mM β-mercaptoethanol, 1 M urea, micrococcal nuclease (New England Biolabs M02474; 1000 Kunitz Units per gram of cell pellet), followed by sonication (15 s on and 15 s off) for a total time of 1 min. Lysates were cleared by centrifugation at 17 500 g for 20 min at 4°C and supernatants were incubated for 1 h with Ni-sepharose beads at 4°C.

    Article Title: N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation
    Article Snippet: .. GST, GST-P, or GST-P3A was expressed in BL21 cells, treated with the lysis buffer provided by a ProFound GST pulldown protein-protein interaction kit (Pierce), and incubated for 30 min. After centrifugation at 13,000 rpm for 30 min, equal amounts of supernatants were mixed with 200 ng purified His-tagged P protein as described by Chen et al. ( ). ..

    Amplification:

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: Production of polyclonal antiserum to gpD The authentic (non-codon-optimized) λ phage gpD gene was amplified by PCR from pAT101 (kindly provided by Dr Andreas Plückthun and Dr Patrick Forrer, University of Zurich, Switzerland) ( , ) using the gpD forward primer (5′-ggtgcc catatg gcgagcaaagaaacctttacc-3′) and the gpD reverse primer (5′-ccgacg ggatcc tcattaaacgatgctgattgc-3′) and then cloned into the pET15b plasmid (Novagen) using the NdeI and BamHI restriction sites (underlined). .. The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen).

    Binding Assay:

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: We added sequences encoding a His6 -MBP (six histidine-maltose binding protein) tag at the N-terminus of FUS, generating the His6 -MBP-FUS construct ( ). .. This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight.

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: Briefly, Guss et al. reported that the C-terminal portion of protein G is responsible for the binding of IgG [ ]. .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden).

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. After the binding of the appropriate proteins to glutathione-Sepharose resins (Amersham Biosciences), the protein-bound resins were incubated with cell lysates overnight at 4°C in immunoprecipitation buffer consisting of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl2 , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 20 mM NaF, and protease inhibitor (complete; Roche).

    Synthesized:

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: As described above, the DNA fragment encoding the spacer and C1 regions (67 residues) was synthesized and used for recombinant protein production. .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden).

    Construct:

    Article Title: Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s
    Article Snippet: Kinesin proteins expression Kinesin proteins were expressed in BL21 cells (BL21(DE3)pLysS, Thermofisher). .. The motor construct (M) includes Drosophila melanogaster KLP10A residues 279–615 and was expressed as previously reported .

    Article Title: When a Module is not a Domain – the Case of the REJ Module and the Redefinition of the Architecture of Polycystin-1
    Article Snippet: .. For protein expression in inclusion bodies constructs were transformed into BL21* cells (Invitrogen). .. Cells were grown at 37 °C and expression was induced with 0.5 mM IPTG (Melford Labs.) at an OD of 0.8 for 4h.

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: We added sequences encoding a His6 -MBP (six histidine-maltose binding protein) tag at the N-terminus of FUS, generating the His6 -MBP-FUS construct ( ). .. This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight.

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: Because residues at the N and C termini of the VA387 P domain structure were disordered , we designed our constructs to omit these regions. .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen).

    Article Title: Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling
    Article Snippet: For protein expression, transformed BL21 cells were grown in minimal medium [Na2 HPO4 , KH2 PO4 , NaCl, MgSO4 , CaCl2 , MEM vitamin mix (Gibco), 15 NH4 Cl, 13 C glucose, Isogrow 15 N 13 C, biotin, and FeCl2 ] with ampicillin. .. Initial 1 HN , 15 N, 13 Cα, 13 Cβ, and 13 C′ backbone resonance assignments of a shorter talin USH-I/LWEQ construct lacking the dimerization helix (2300–2482) were made at 318 K and pH 6.0 using standard 3D triple-resonance experiments ( ).

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: The fusion protein was constructed using the parent vector pET28b at the Nco I/ Sal I insertion site. .. The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD).

    Incubation:

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. .. Bacterial cells were pelleted at 6000 rpm for 10 min and lysed in lysis buffer (1 M KCl, 50 mM Tris pH 7.4, 10 mM imidazole, 1 mM CaCl2 , 5% glycerol, 1% NP40, 1.5 mM β-mercaptoethanol, 1 M urea, micrococcal nuclease (New England Biolabs M02474; 1000 Kunitz Units per gram of cell pellet), followed by sonication (15 s on and 15 s off) for a total time of 1 min. Lysates were cleared by centrifugation at 17 500 g for 20 min at 4°C and supernatants were incubated for 1 h with Ni-sepharose beads at 4°C.

    Article Title: N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation
    Article Snippet: .. GST, GST-P, or GST-P3A was expressed in BL21 cells, treated with the lysis buffer provided by a ProFound GST pulldown protein-protein interaction kit (Pierce), and incubated for 30 min. After centrifugation at 13,000 rpm for 30 min, equal amounts of supernatants were mixed with 200 ng purified His-tagged P protein as described by Chen et al. ( ). ..

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. After the binding of the appropriate proteins to glutathione-Sepharose resins (Amersham Biosciences), the protein-bound resins were incubated with cell lysates overnight at 4°C in immunoprecipitation buffer consisting of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl2 , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 20 mM NaF, and protease inhibitor (complete; Roche).

    Article Title: Cardioprotective Effect of Nicorandil, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Prolongs Survival in HSPB5 R120G Transgenic Mice
    Article Snippet: Recombinant protein To produce a recombinant protein, His epitope-tagged wild-type HSPB5-FLAG, HSPB5 R120G-FLAG were overexpressed in BL21 cells (Invitrogen, Carlsbad, CA, USA) using the pET system (Novagen, Madison, WI) and purified with a Ni-NTA column (Qiagen, Santa Clarita, CA) as described previously . .. The amyloid oligomer level, each recombinant protein or protein mixture with nicorandil was incubated and blotted on a nitrocellulose membrane and quantified as described previously .

    Expressing:

    Article Title: Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s
    Article Snippet: .. Kinesin proteins expression Kinesin proteins were expressed in BL21 cells (BL21(DE3)pLysS, Thermofisher). .. The motor construct (M) includes Drosophila melanogaster KLP10A residues 279–615 and was expressed as previously reported .

    Article Title: When a Module is not a Domain – the Case of the REJ Module and the Redefinition of the Architecture of Polycystin-1
    Article Snippet: .. For protein expression in inclusion bodies constructs were transformed into BL21* cells (Invitrogen). .. Cells were grown at 37 °C and expression was induced with 0.5 mM IPTG (Melford Labs.) at an OD of 0.8 for 4h.

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: .. This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. ..

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden). .. Generation of an Antiserum for Mouse CD146 and 4D5-Fc Antibodies Total RNA was isolated from the B16-F10 cells and used for cDNA synthesis.

    Article Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery
    Article Snippet: Purification of TrwBΔN70/VirD4ΔN70 for raising antibodies Overnight culture of a BL21* cells (Thermo Fisher) freshly transformed with pBADM11His_TEV trwB /virD4ΔN70 ( ) was diluted into six litre of LB medium supplemented with appropriate antibiotic. .. When the OD600 of the cell culture reached 0.5, cells were induced by the addition of 0.08% arabinose and the expression was carried out at 16°C with shaking overnight.

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen). .. Expression was induced with IPTG (isopropyl-β- d -thiogalactopyranoside; 1 mM) for 18 h at 22°C.

    Article Title: Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling
    Article Snippet: .. For protein expression, transformed BL21 cells were grown in minimal medium [Na2 HPO4 , KH2 PO4 , NaCl, MgSO4 , CaCl2 , MEM vitamin mix (Gibco), 15 NH4 Cl, 13 C glucose, Isogrow 15 N 13 C, biotin, and FeCl2 ] with ampicillin. .. GST fusion proteins with an intervening PreScission protease recognition site were expressed and purified as described , cleaved on-column with PreScission protease, eluted, and concentrated to 1 mM.

    Article Title: An Undecided Coiled Coil
    Article Snippet: .. Protein Expression and Purification For protein expression, miniprep DNA was transformed into BL21* cells (Invitrogen). .. Transformed cells were grown up in LB medium to OD ∼0.8 when they were induced with 0.75 mm IPTG for 4 h. For 15 N isotope labeling the protocol was modified as suggested ( ).

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: Paragraph title: Protein expression and antibody production ... The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen.

    Modification:

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: The Fc-G67 polypeptide was produced for modification of the anti-HER2 antibody (4D5-Fc) based on previous reports [ ]. .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden).

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen). .. Expression was induced with IPTG (isopropyl-β- d -thiogalactopyranoside; 1 mM) for 18 h at 22°C.

    Article Title: An Undecided Coiled Coil
    Article Snippet: Protein Expression and Purification For protein expression, miniprep DNA was transformed into BL21* cells (Invitrogen). .. Transformed cells were grown up in LB medium to OD ∼0.8 when they were induced with 0.75 mm IPTG for 4 h. For 15 N isotope labeling the protocol was modified as suggested ( ).

    Western Blot:

    Article Title: N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation
    Article Snippet: GST, GST-P, or GST-P3A was expressed in BL21 cells, treated with the lysis buffer provided by a ProFound GST pulldown protein-protein interaction kit (Pierce), and incubated for 30 min. After centrifugation at 13,000 rpm for 30 min, equal amounts of supernatants were mixed with 200 ng purified His-tagged P protein as described by Chen et al. ( ). .. Proteins in supernatants and eluted proteins from GST pulldown beads were detected by Western blotting with anti-HA, anti-GST, or anti-N Ab.

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD). .. Serum of final bleeds recognized a 116-kD protein in immunoblots of microsomal proteins from ECA1 transformants.

    Transformation Assay:

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: .. The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen). .. Transformed bacteria were grown to mid-log phase and then induced for 3 h with isopropyl-β-d -thiogalactopyranoside (IPTG) added to a final concentration of 1 mM.

    Article Title: When a Module is not a Domain – the Case of the REJ Module and the Redefinition of the Architecture of Polycystin-1
    Article Snippet: .. For protein expression in inclusion bodies constructs were transformed into BL21* cells (Invitrogen). .. Cells were grown at 37 °C and expression was induced with 0.5 mM IPTG (Melford Labs.) at an OD of 0.8 for 4h.

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: .. This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. ..

    Article Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery
    Article Snippet: .. Purification of TrwBΔN70/VirD4ΔN70 for raising antibodies Overnight culture of a BL21* cells (Thermo Fisher) freshly transformed with pBADM11His_TEV trwB /virD4ΔN70 ( ) was diluted into six litre of LB medium supplemented with appropriate antibiotic. .. When the OD600 of the cell culture reached 0.5, cells were induced by the addition of 0.08% arabinose and the expression was carried out at 16°C with shaking overnight.

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen). .. Expression was induced with IPTG (isopropyl-β- d -thiogalactopyranoside; 1 mM) for 18 h at 22°C.

    Article Title: Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling
    Article Snippet: .. For protein expression, transformed BL21 cells were grown in minimal medium [Na2 HPO4 , KH2 PO4 , NaCl, MgSO4 , CaCl2 , MEM vitamin mix (Gibco), 15 NH4 Cl, 13 C glucose, Isogrow 15 N 13 C, biotin, and FeCl2 ] with ampicillin. .. GST fusion proteins with an intervening PreScission protease recognition site were expressed and purified as described , cleaved on-column with PreScission protease, eluted, and concentrated to 1 mM.

    Article Title: An Undecided Coiled Coil
    Article Snippet: .. Protein Expression and Purification For protein expression, miniprep DNA was transformed into BL21* cells (Invitrogen). .. Transformed cells were grown up in LB medium to OD ∼0.8 when they were induced with 0.75 mm IPTG for 4 h. For 15 N isotope labeling the protocol was modified as suggested ( ).

    Crystallization Assay:

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: Paragraph title: Protein expression, purification, and crystallization of the norovirus P domain. ... The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen).

    Sequencing:

    Article Title: Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s
    Article Snippet: Kinesin proteins expression Kinesin proteins were expressed in BL21 cells (BL21(DE3)pLysS, Thermofisher). .. A TEV protease site (ENLYFQG) was introduced between the His tag and the start of the KLP10A sequence.

    Article Title: When a Module is not a Domain – the Case of the REJ Module and the Redefinition of the Architecture of Polycystin-1
    Article Snippet: The constructs are expressed as fusion protein with the sequence MHHHHHHSSGVDLGTENLYFQSM, containing a his-tag and a TEV site, N-terminally attached which adds 23 residues and 2.7 kD to each domain. .. For protein expression in inclusion bodies constructs were transformed into BL21* cells (Invitrogen).

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: A 55-amino-acid sequence is repeated three times in the C1, C2, and C3 regions and binds to IgG. .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden).

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: 2.5 Protein expression and antibody production A 701 bp chicken LRP2 cDNA fragment encoding the intracellular domain ( A) with a C-terminal 6xHis-tag was generated by RT-PCR using the following oligonucleotides: forward, 5′- GAA TTC GGA GTG TTA GCG ATT GGA GGC-3′ (EcoRI restriction site in boldface) and reverse, 5′- GCG GCC GCT TAA TGA TGA TGA TGA TGA TGA TGC TCT TTA ACA AGA TTG GCG GTG-3′ (NotI restriction site in boldface, and 6xHis-tag sequence in italics ). .. The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen.

    Chromatography:

    Article Title: The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo
    Article Snippet: .. Protein was expressed in BL21 cells (Invitrogen), and purified using Ni-NTA chromatography (QIAGEN). .. Purified protein was given to Bioklone, Chennai, India to generate monoclonal antibodies.

    Concentration Assay:

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen). .. Transformed bacteria were grown to mid-log phase and then induced for 3 h with isopropyl-β-d -thiogalactopyranoside (IPTG) added to a final concentration of 1 mM.

    Protease Inhibitor:

    Article Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery
    Article Snippet: Purification of TrwBΔN70/VirD4ΔN70 for raising antibodies Overnight culture of a BL21* cells (Thermo Fisher) freshly transformed with pBADM11His_TEV trwB /virD4ΔN70 ( ) was diluted into six litre of LB medium supplemented with appropriate antibiotic. .. Cells were pelleted by spinning down for 30 min at 5,000 × g and resuspended in lysis buffer: 50 mM HEPES 7.6, 250 mM NaCl, 1 mM EDTA with protease inhibitor cocktail tablet (Roche), 100 μg/ml lysozyme and 1 μg/ml DNaseI.

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. After the binding of the appropriate proteins to glutathione-Sepharose resins (Amersham Biosciences), the protein-bound resins were incubated with cell lysates overnight at 4°C in immunoprecipitation buffer consisting of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl2 , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 20 mM NaF, and protease inhibitor (complete; Roche).

    Cell Culture:

    Article Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery
    Article Snippet: Purification of TrwBΔN70/VirD4ΔN70 for raising antibodies Overnight culture of a BL21* cells (Thermo Fisher) freshly transformed with pBADM11His_TEV trwB /virD4ΔN70 ( ) was diluted into six litre of LB medium supplemented with appropriate antibiotic. .. When the OD600 of the cell culture reached 0.5, cells were induced by the addition of 0.08% arabinose and the expression was carried out at 16°C with shaking overnight.

    SDS Page:

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. The resins were then washed five times, and resin-bound proteins were eluted and resolved by SDS-PAGE and were detected by immunoblotting with anti-Tax antibody.

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: .. The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD). .. The purified fusion protein samples were injected into New Zealand white rabbits with RIBI adjuvant as recommended by the manufacturer (RIBI ImmunoChem Research).

    Generated:

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: 2.5 Protein expression and antibody production A 701 bp chicken LRP2 cDNA fragment encoding the intracellular domain ( A) with a C-terminal 6xHis-tag was generated by RT-PCR using the following oligonucleotides: forward, 5′- GAA TTC GGA GTG TTA GCG ATT GGA GGC-3′ (EcoRI restriction site in boldface) and reverse, 5′- GCG GCC GCT TAA TGA TGA TGA TGA TGA TGA TGC TCT TTA ACA AGA TTG GCG GTG-3′ (NotI restriction site in boldface, and 6xHis-tag sequence in italics ). .. The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: 2.5 Protein expression and antibody production A 701 bp chicken LRP2 cDNA fragment encoding the intracellular domain ( A) with a C-terminal 6xHis-tag was generated by RT-PCR using the following oligonucleotides: forward, 5′- GAA TTC GGA GTG TTA GCG ATT GGA GGC-3′ (EcoRI restriction site in boldface) and reverse, 5′- GCG GCC GCT TAA TGA TGA TGA TGA TGA TGA TGC TCT TTA ACA AGA TTG GCG GTG-3′ (NotI restriction site in boldface, and 6xHis-tag sequence in italics ). .. The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen.

    Sonication:

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. .. Bacterial cells were pelleted at 6000 rpm for 10 min and lysed in lysis buffer (1 M KCl, 50 mM Tris pH 7.4, 10 mM imidazole, 1 mM CaCl2 , 5% glycerol, 1% NP40, 1.5 mM β-mercaptoethanol, 1 M urea, micrococcal nuclease (New England Biolabs M02474; 1000 Kunitz Units per gram of cell pellet), followed by sonication (15 s on and 15 s off) for a total time of 1 min. Lysates were cleared by centrifugation at 17 500 g for 20 min at 4°C and supernatants were incubated for 1 h with Ni-sepharose beads at 4°C.

    Affinity Purification:

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: The fusion proteins were expressed in Escherichia coli DH10α, and affinity purified over a glutathione-agarose column (Pharmacia Biotech, Piscataway, NJ). .. The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD).

    Recombinant:

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden). .. Generation of an Antiserum for Mouse CD146 and 4D5-Fc Antibodies Total RNA was isolated from the B16-F10 cells and used for cDNA synthesis.

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: .. Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. After the binding of the appropriate proteins to glutathione-Sepharose resins (Amersham Biosciences), the protein-bound resins were incubated with cell lysates overnight at 4°C in immunoprecipitation buffer consisting of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl2 , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 20 mM NaF, and protease inhibitor (complete; Roche).

    Article Title: Cardioprotective Effect of Nicorandil, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Prolongs Survival in HSPB5 R120G Transgenic Mice
    Article Snippet: .. Recombinant protein To produce a recombinant protein, His epitope-tagged wild-type HSPB5-FLAG, HSPB5 R120G-FLAG were overexpressed in BL21 cells (Invitrogen, Carlsbad, CA, USA) using the pET system (Novagen, Madison, WI) and purified with a Ni-NTA column (Qiagen, Santa Clarita, CA) as described previously . .. The amyloid oligomer level, each recombinant protein or protein mixture with nicorandil was incubated and blotted on a nitrocellulose membrane and quantified as described previously .

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen. .. Antiserum against recombinant ggLRP2 was raised in adult female New Zealand White rabbits by injections of antigen, 200 mg each, as described previously ( ).

    Pull Down Assay:

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: Paragraph title: GST pull-down assay. ... Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside.

    Isolation:

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: The norovirus Hiro GII.12 strain (GenBank accession number ) was isolated from an adult male in a small outbreak of gastroenteritis in November 1999 in Hiroshima, Japan ( ). .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen).

    Size-exclusion Chromatography:

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen). .. The P domain was further purified by size exclusion chromatography with a Superdex-200 column (GE), concentrated to 2 to 10 mg/ml, and stored in GFB (0.35 M NaCl, 2.5 mM Tris [pH 7.0], 0.02% NaN3 ) before crystallization.

    Labeling:

    Article Title: An Undecided Coiled Coil
    Article Snippet: Protein Expression and Purification For protein expression, miniprep DNA was transformed into BL21* cells (Invitrogen). .. Transformed cells were grown up in LB medium to OD ∼0.8 when they were induced with 0.75 mm IPTG for 4 h. For 15 N isotope labeling the protocol was modified as suggested ( ).

    Purification:

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen). .. Thawed lysate was purified using a Co2+ column (BD Talon) according to the manufacturer's recommendations.

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: Paragraph title: Protein expression and purification ... This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight.

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden). .. Generation of an Antiserum for Mouse CD146 and 4D5-Fc Antibodies Total RNA was isolated from the B16-F10 cells and used for cDNA synthesis.

    Article Title: N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation
    Article Snippet: .. GST, GST-P, or GST-P3A was expressed in BL21 cells, treated with the lysis buffer provided by a ProFound GST pulldown protein-protein interaction kit (Pierce), and incubated for 30 min. After centrifugation at 13,000 rpm for 30 min, equal amounts of supernatants were mixed with 200 ng purified His-tagged P protein as described by Chen et al. ( ). ..

    Article Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery
    Article Snippet: .. Purification of TrwBΔN70/VirD4ΔN70 for raising antibodies Overnight culture of a BL21* cells (Thermo Fisher) freshly transformed with pBADM11His_TEV trwB /virD4ΔN70 ( ) was diluted into six litre of LB medium supplemented with appropriate antibiotic. .. When the OD600 of the cell culture reached 0.5, cells were induced by the addition of 0.08% arabinose and the expression was carried out at 16°C with shaking overnight.

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: Paragraph title: Protein expression, purification, and crystallization of the norovirus P domain. ... The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen).

    Article Title: Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling
    Article Snippet: For protein expression, transformed BL21 cells were grown in minimal medium [Na2 HPO4 , KH2 PO4 , NaCl, MgSO4 , CaCl2 , MEM vitamin mix (Gibco), 15 NH4 Cl, 13 C glucose, Isogrow 15 N 13 C, biotin, and FeCl2 ] with ampicillin. .. GST fusion proteins with an intervening PreScission protease recognition site were expressed and purified as described , cleaved on-column with PreScission protease, eluted, and concentrated to 1 mM.

    Article Title: An Undecided Coiled Coil
    Article Snippet: .. Protein Expression and Purification For protein expression, miniprep DNA was transformed into BL21* cells (Invitrogen). .. Transformed cells were grown up in LB medium to OD ∼0.8 when they were induced with 0.75 mm IPTG for 4 h. For 15 N isotope labeling the protocol was modified as suggested ( ).

    Article Title: The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo
    Article Snippet: .. Protein was expressed in BL21 cells (Invitrogen), and purified using Ni-NTA chromatography (QIAGEN). .. Purified protein was given to Bioklone, Chennai, India to generate monoclonal antibodies.

    Article Title: Cardioprotective Effect of Nicorandil, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Prolongs Survival in HSPB5 R120G Transgenic Mice
    Article Snippet: .. Recombinant protein To produce a recombinant protein, His epitope-tagged wild-type HSPB5-FLAG, HSPB5 R120G-FLAG were overexpressed in BL21 cells (Invitrogen, Carlsbad, CA, USA) using the pET system (Novagen, Madison, WI) and purified with a Ni-NTA column (Qiagen, Santa Clarita, CA) as described previously . .. The amyloid oligomer level, each recombinant protein or protein mixture with nicorandil was incubated and blotted on a nitrocellulose membrane and quantified as described previously .

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: .. The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen. .. Antiserum against recombinant ggLRP2 was raised in adult female New Zealand White rabbits by injections of antigen, 200 mg each, as described previously ( ).

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: .. The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD). .. The purified fusion protein samples were injected into New Zealand white rabbits with RIBI adjuvant as recommended by the manufacturer (RIBI ImmunoChem Research).

    Polymerase Chain Reaction:

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: Production of polyclonal antiserum to gpD The authentic (non-codon-optimized) λ phage gpD gene was amplified by PCR from pAT101 (kindly provided by Dr Andreas Plückthun and Dr Patrick Forrer, University of Zurich, Switzerland) ( , ) using the gpD forward primer (5′-ggtgcc catatg gcgagcaaagaaacctttacc-3′) and the gpD reverse primer (5′-ccgacg ggatcc tcattaaacgatgctgattgc-3′) and then cloned into the pET15b plasmid (Novagen) using the NdeI and BamHI restriction sites (underlined). .. The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen).

    Lysis:

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. .. Bacterial cells were pelleted at 6000 rpm for 10 min and lysed in lysis buffer (1 M KCl, 50 mM Tris pH 7.4, 10 mM imidazole, 1 mM CaCl2 , 5% glycerol, 1% NP40, 1.5 mM β-mercaptoethanol, 1 M urea, micrococcal nuclease (New England Biolabs M02474; 1000 Kunitz Units per gram of cell pellet), followed by sonication (15 s on and 15 s off) for a total time of 1 min. Lysates were cleared by centrifugation at 17 500 g for 20 min at 4°C and supernatants were incubated for 1 h with Ni-sepharose beads at 4°C.

    Article Title: N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation
    Article Snippet: .. GST, GST-P, or GST-P3A was expressed in BL21 cells, treated with the lysis buffer provided by a ProFound GST pulldown protein-protein interaction kit (Pierce), and incubated for 30 min. After centrifugation at 13,000 rpm for 30 min, equal amounts of supernatants were mixed with 200 ng purified His-tagged P protein as described by Chen et al. ( ). ..

    Article Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery
    Article Snippet: Purification of TrwBΔN70/VirD4ΔN70 for raising antibodies Overnight culture of a BL21* cells (Thermo Fisher) freshly transformed with pBADM11His_TEV trwB /virD4ΔN70 ( ) was diluted into six litre of LB medium supplemented with appropriate antibiotic. .. Cells were pelleted by spinning down for 30 min at 5,000 × g and resuspended in lysis buffer: 50 mM HEPES 7.6, 250 mM NaCl, 1 mM EDTA with protease inhibitor cocktail tablet (Roche), 100 μg/ml lysozyme and 1 μg/ml DNaseI.

    Injection:

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD). .. The purified fusion protein samples were injected into New Zealand white rabbits with RIBI adjuvant as recommended by the manufacturer (RIBI ImmunoChem Research).

    Plasmid Preparation:

    Article Title: Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s
    Article Snippet: Kinesin proteins expression Kinesin proteins were expressed in BL21 cells (BL21(DE3)pLysS, Thermofisher). .. The neck motor construct (NM) includes KLP10A residues 198–615 cloned into pRSET B plasmid with an N-terminal histidine tag.

    Article Title: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda
    Article Snippet: .. The resulting pET15b-gpD plasmid was transformed into BL21 cells (Invitrogen). .. Transformed bacteria were grown to mid-log phase and then induced for 3 h with isopropyl-β-d -thiogalactopyranoside (IPTG) added to a final concentration of 1 mM.

    Article Title: Nucleic acid-binding specificity of human FUS protein
    Article Snippet: .. This expression plasmid was transformed into BL21 cells (Life Technologies) and grown in a 5-ml LB-Amp culture overnight. ..

    Article Title: Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability ▿ †Crystal Structures of GII.10 and GII.12 Norovirus Protruding
    Article Snippet: .. The near-full-length GII.10 (residues 224 to 538) and GII.12 (residues 224 to 525) P domains (314 and 301 amino acids in length, respectively) were optimized for Escherichia coli expression, cloned in a modified pMal-c2x vector at BamHI and NotI (New England Biolabs), and transformed into BL21 cells (Invitrogen). .. Expression was induced with IPTG (isopropyl-β- d -thiogalactopyranoside; 1 mM) for 18 h at 22°C.

    Article Title: The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo
    Article Snippet: Monoclonal antibody generation The protein region of UNC-104 (amino acid 740-1117) was cloned into pRSETA vector (Invitrogen) using standard techniques. .. Protein was expressed in BL21 cells (Invitrogen), and purified using Ni-NTA chromatography (QIAGEN).

    Article Title: Renal LRP2 expression in man and chicken is estrogen-responsive
    Article Snippet: The fragment was cloned into the pGEX-5X-1 expression vector to provide an N-terminal GST-tag. .. The cloned ggGST-LRP2-His-construct was expressed in BL21 cells (Invitrogen) and purified using Ni-NTA beads technology from QIAgen.

    Article Title: An Endoplasmic Reticulum-Bound Ca2+/Mn2+ Pump, ECA1, Supports Plant Growth and Confers Tolerance to Mn2+ Stress 1
    Article Snippet: The fusion protein was constructed using the parent vector pET28b at the Nco I/ Sal I insertion site. .. The fusion protein was expressed in BL21 cells, and first purified through Probond nickel-chelating resin according to the manufacturer's protocol (Invitrogen Company Xpress System, Invitrogen, Carlsbad, CA) and purified by SDS-PAGE (approximately 25 kD).

    Positron Emission Tomography:

    Article Title: Cardioprotective Effect of Nicorandil, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Prolongs Survival in HSPB5 R120G Transgenic Mice
    Article Snippet: .. Recombinant protein To produce a recombinant protein, His epitope-tagged wild-type HSPB5-FLAG, HSPB5 R120G-FLAG were overexpressed in BL21 cells (Invitrogen, Carlsbad, CA, USA) using the pET system (Novagen, Madison, WI) and purified with a Ni-NTA column (Qiagen, Santa Clarita, CA) as described previously . .. The amyloid oligomer level, each recombinant protein or protein mixture with nicorandil was incubated and blotted on a nitrocellulose membrane and quantified as described previously .

    Nuclear Magnetic Resonance:

    Article Title: Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling
    Article Snippet: Paragraph title: NMR. ... For protein expression, transformed BL21 cells were grown in minimal medium [Na2 HPO4 , KH2 PO4 , NaCl, MgSO4 , CaCl2 , MEM vitamin mix (Gibco), 15 NH4 Cl, 13 C glucose, Isogrow 15 N 13 C, biotin, and FeCl2 ] with ampicillin.

    Produced:

    Article Title: Development of Antibody-Modified Nanobubbles Using Fc-Region-Binding Polypeptides for Ultrasound Imaging
    Article Snippet: .. The recombinant proteins were produced in BL21 cells (Thermo Fisher Scientific, Waltham, MA, USA) using MagicMedia™ E. coli expression medium (Thermo Fisher Scientific, Waltham, MA, USA) and were purified with a glutathione sepharose column (GE Healthcare Bio-Sciences, Uppsala, Sweden). .. Generation of an Antiserum for Mouse CD146 and 4D5-Fc Antibodies Total RNA was isolated from the B16-F10 cells and used for cDNA synthesis.

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: .. Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. After the binding of the appropriate proteins to glutathione-Sepharose resins (Amersham Biosciences), the protein-bound resins were incubated with cell lysates overnight at 4°C in immunoprecipitation buffer consisting of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl2 , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 20 mM NaF, and protease inhibitor (complete; Roche).

    Immunoprecipitation:

    Article Title: Peptidylproline cis-trans-Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-?B ▿
    Article Snippet: Recombinant GST and GST-Pin1 were produced in BL21 cells (Invitrogen) following treatment of cells with 1 mM isopropyl-β- d -thiogalactopyranoside. .. After the binding of the appropriate proteins to glutathione-Sepharose resins (Amersham Biosciences), the protein-bound resins were incubated with cell lysates overnight at 4°C in immunoprecipitation buffer consisting of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl2 , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 20 mM NaF, and protease inhibitor (complete; Roche).

    Staining:

    Article Title: The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo
    Article Snippet: Protein was expressed in BL21 cells (Invitrogen), and purified using Ni-NTA chromatography (QIAGEN). .. All monoclonal antibodies tested showed pan-neural staining in wild type animals and no staining in the unc-104(rh142) animals ( ).

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    Thermo Fisher e coli bl21 cells
    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced <t>BL21</t> (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.
    E Coli Bl21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21 cells/product/Thermo Fisher
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    e coli bl21 cells - by Bioz Stars, 2020-02
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    79
    Thermo Fisher e coli bl21 expressing gex4t1 tarbp2 wt
    <t>TARBP2</t> is mainly SUMOylated at K52. ( a ) Mutation K52R abolishes TARBP2 SUMOylation in 293T cells. pLPC-TARBP2-WT or -K52R with His-SUMO1/Flag-Ubc9 was co-transfected into 293T cells. Forty-eight hours after transfection, cells were lysed and Ni 2+ -NTA pull down was performed to detect TARBP2 SUMOylation. ( b ) Mutation K52R impairs TARBP2 SUMOylation in an E. coli system. Plasmid pGEX4T1-TARBP2-WT or -K52R with pE1E2S1 plasmid were co-transfected into E. coli <t>BL21</t> (DE3). Western blot analysis was conducted with anti-SUMO1 antibody after GST pull down, and the same membrane was also detected with anti-TARBP2 antibody after stripping. ( c ) Hypoxia downregulates SUMOylation of TARBP2. 293T cells transfected with indicated plasmids were cultured under 1% of oxygen condition (hypoxia) for indicated time before being harvested. Ni 2+ -NTA resin pull down was performed to detect SUMOylated TARBP2. ( d ) Hydrogen peroxide (H 2 O 2 ) upregulates SUMOylation of TARBP2. 293T cells transfected with indicated plasmids were treated with 100 μM of H 2 O 2 for indicated time before being harvested. Ni 2+ -NTA resin pull down was performed to detect SUMOylated TARBP2. For full scans of western blots ( a – d ), see Supplementary Fig. 8 .
    E Coli Bl21 Expressing Gex4t1 Tarbp2 Wt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher e coli bl21 competent cells
    Effects of recombinant TPY SufS on the growth of E. coli <t>BL21</t> in the absence and the presence of sodium thiosulfate. a The variation of cell density of E. coli BL21 cells in which recombinant TPY SufS was over-expressed in vivo; b the variations of cell density of E. coli BL21 when directly adding purified TPY SufS (520 ng/mL) and actin protein (520 ng/mL) into medium; c the variation of cell density of E. coli BL21 with over-expression of recombinant TPY SufS in the presence of sodium thiosulfate of 6% (w/v)
    E Coli Bl21 Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Journal: Oncotarget

    Article Title: Identification and screening of effective protective antigens for channel catfish against Streptococcus iniae

    doi: 10.18632/oncotarget.16475

    Figure Lengend Snippet: Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Article Snippet: The plasmids were then transformed into E. coli BL21 cells and induced by adding 1.0 mM IPTG at 37°C for 4 h. The cells were then centrifuged at 8000 × g for 10 min at 4°C, suspended in sterile phosphate-buffered saline (PBS), sonicated with a Sonic Dismembrator (model 500; Thermo Fisher Scientific, Waltham, MA, USA), and examined by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

    Techniques: Recombinant, Expressing, Purification, Marker, Polymerase Chain Reaction

    Spacer-seq identifies hundreds of off-target spacer integration sites within the E. coli genome a , Schematic of the Spacer-seq workflow. Fragmentation of isolated genomic DNA containing DSA events (step 1). Ligation of adaptor sequences onto fragment ends (step 2). PCR amplification using the defined spacer sequence and adaptor sequence as primers (step 3) for specific enrichment of fragments containing spacer insertions (step 4). High-throughput sequencing of enriched fragments and mapping of reads to the reference genome (step 5). b , The diagram of the genome shows an example of a single Spacer-seq experiment with the number of reads mapped to the E. coli BL21 genome (binned per 10 kb). Dashed lines represent 100 reads. c , The percentage of Spacer-seq reads mapped to a CRISPR array, or to off-target sites within the genome or expression plasmid. Error bars represent mean ± s.d., n = 3 biological replicates. d , Comparison between the average number of off-target integration events mapped to the genome or plasmid, normalized by the total DNA content within the cell (assuming ~30 plasmids per cell). Error bars represent mean ± s.d., n = 3 biological replicates. e , WebLogo of the ~700 unique off-target integration sites identified by Spacer-seq, aligned to the BL21 CRISPR1 array leader and the repeat sequence. f , The percentage of expanded arrays after the DSA experiment. Plasmid containing the minimal version of the K12 CRISPR1 array (native repeat) is compared to a mutant version with repeat mutations C14G and A15C (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. * P = 0.04 calculated with a two-sample unpaired t -test. g , The percentage of expanded arrays after the DSA experiment. The genomic CRISPR1 array (native repeat) is compared to a strain in which the entire CRISPR1 locus is replaced with a minimal array consisting of a 100-nt leader and a single mutant repeat (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. ** P = 0.002 calculated with a two-sample unpaired t -test. Open circles represent individual replicate data points.

    Journal: Nature microbiology

    Article Title: Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Spacer-seq identifies hundreds of off-target spacer integration sites within the E. coli genome a , Schematic of the Spacer-seq workflow. Fragmentation of isolated genomic DNA containing DSA events (step 1). Ligation of adaptor sequences onto fragment ends (step 2). PCR amplification using the defined spacer sequence and adaptor sequence as primers (step 3) for specific enrichment of fragments containing spacer insertions (step 4). High-throughput sequencing of enriched fragments and mapping of reads to the reference genome (step 5). b , The diagram of the genome shows an example of a single Spacer-seq experiment with the number of reads mapped to the E. coli BL21 genome (binned per 10 kb). Dashed lines represent 100 reads. c , The percentage of Spacer-seq reads mapped to a CRISPR array, or to off-target sites within the genome or expression plasmid. Error bars represent mean ± s.d., n = 3 biological replicates. d , Comparison between the average number of off-target integration events mapped to the genome or plasmid, normalized by the total DNA content within the cell (assuming ~30 plasmids per cell). Error bars represent mean ± s.d., n = 3 biological replicates. e , WebLogo of the ~700 unique off-target integration sites identified by Spacer-seq, aligned to the BL21 CRISPR1 array leader and the repeat sequence. f , The percentage of expanded arrays after the DSA experiment. Plasmid containing the minimal version of the K12 CRISPR1 array (native repeat) is compared to a mutant version with repeat mutations C14G and A15C (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. * P = 0.04 calculated with a two-sample unpaired t -test. g , The percentage of expanded arrays after the DSA experiment. The genomic CRISPR1 array (native repeat) is compared to a strain in which the entire CRISPR1 locus is replaced with a minimal array consisting of a 100-nt leader and a single mutant repeat (OTCR). Error bars represent mean ± s.e.m. n = 3 biological replicates. ** P = 0.002 calculated with a two-sample unpaired t -test. Open circles represent individual replicate data points.

    Article Snippet: Briefly, liquid cultures of E. coli BL21-AI cells (Thermo) harbouring a plasmid expressing Cas1 and Cas2 under the control of a T7-lac promoter (pWUR 1 + 2, which was a generous gift from U. Qimron) were started from plates and grown overnight in LB.

    Techniques: Isolation, Ligation, Polymerase Chain Reaction, Amplification, Sequencing, Next-Generation Sequencing, CRISPR, Expressing, Plasmid Preparation, Mutagenesis

    Effects of genomic knockouts of IHF and the CRISPR1 locus on off-target spacer integration activity a , The percentage of oligo integrations into the CRISPR1 locus or the off-target sites normalized per cell (array) following DSA in the BL21-AI strain (WT), or the BL21-AI strain with either the IHF-α or the IHF-β subunits knocked out ΔIHF-α and ΔIHF-β, respectively). Error bars represent mean ± s.d. b , The percentage of Spacer-seq reads aligned on-target to the CRISPR1 locus or to other regions in the genome (off-target) in the WT, ΔIHF-α, and ΔIHF-β strains. Error bars represent mean ± s.d. c , Pearson correlation coefficient (r) of the off-target site identities between the WT versus ΔIHF-α/β strain, WT versus CRISPR1 deletion (ΔCRISPRI) strain and WT versus WT replicates. Error bars represent mean ± s.d. Open circles represent individual replicate data points. d , The percentage of Spacer-seq reads aligned to unique off-target sites within the genome. The knockout strain percentages ( y axis) of the ΔIHF-α/β strains and the ΔCRISPR1 strain are compared to those of WT ( x axis). Each point represents a unique genomic site. For all panels, n = 4 (for WT) and n = 3 (for knockout strains) biological replicates.

    Journal: Nature microbiology

    Article Title: Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Effects of genomic knockouts of IHF and the CRISPR1 locus on off-target spacer integration activity a , The percentage of oligo integrations into the CRISPR1 locus or the off-target sites normalized per cell (array) following DSA in the BL21-AI strain (WT), or the BL21-AI strain with either the IHF-α or the IHF-β subunits knocked out ΔIHF-α and ΔIHF-β, respectively). Error bars represent mean ± s.d. b , The percentage of Spacer-seq reads aligned on-target to the CRISPR1 locus or to other regions in the genome (off-target) in the WT, ΔIHF-α, and ΔIHF-β strains. Error bars represent mean ± s.d. c , Pearson correlation coefficient (r) of the off-target site identities between the WT versus ΔIHF-α/β strain, WT versus CRISPR1 deletion (ΔCRISPRI) strain and WT versus WT replicates. Error bars represent mean ± s.d. Open circles represent individual replicate data points. d , The percentage of Spacer-seq reads aligned to unique off-target sites within the genome. The knockout strain percentages ( y axis) of the ΔIHF-α/β strains and the ΔCRISPR1 strain are compared to those of WT ( x axis). Each point represents a unique genomic site. For all panels, n = 4 (for WT) and n = 3 (for knockout strains) biological replicates.

    Article Snippet: Briefly, liquid cultures of E. coli BL21-AI cells (Thermo) harbouring a plasmid expressing Cas1 and Cas2 under the control of a T7-lac promoter (pWUR 1 + 2, which was a generous gift from U. Qimron) were started from plates and grown overnight in LB.

    Techniques: Immunohistofluorescence, Activity Assay, Knock-Out

    Comparison of three different NCA sequences and their activity in target interference and primed acquisition a , Schematic of the plasmid-based NCAs that were used in the primed acquisition assays. The arrays contain an inducible promoter that drives the expression of 60 nt of the off-target leader (leader NCA ) along with a 33-nt spacer that matches the M13 bacteriophage genome (spacer M13 ) that is flanked by the 28-nt off-target repeat sequences (repeat NCA ). b , Multiple sequence alignment of the NCA repeats aligned to the BL21 CRISPR repeat sequence. Residues conserved with the BL21 repeat are shown in black. c , Results of the plasmid interference assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing the M13 spacer, or a strain with no plasmid-based array. n = 4 biological replicates (circles). Error bars represent mean ± s.d. c.f.u., colony-forming unit. d , Results of the primed acquisition assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing strains. Black bars correspond to the percentage of total arrays containing new M13-derived spacers. The red bars correspond to the percentage of newly expanded arrays containing M13-derived spacers. n = 3 biological replicates. Error bars represent mean ± s.d. e , Comparison of plasmid-based NCA expansion frequencies following DSA. Expansion frequencies for each NCA were quantified by high-throughput sequencing of the plasmid-based arrays. Each point represents the percentage of expansions detected for each array. We did not detect any expansions for NCAs that do not display a point (fic, potG and yfic) , indicating integration efficiencies of

    Journal: Nature microbiology

    Article Title: Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

    doi: 10.1038/s41564-017-0097-z

    Figure Lengend Snippet: Comparison of three different NCA sequences and their activity in target interference and primed acquisition a , Schematic of the plasmid-based NCAs that were used in the primed acquisition assays. The arrays contain an inducible promoter that drives the expression of 60 nt of the off-target leader (leader NCA ) along with a 33-nt spacer that matches the M13 bacteriophage genome (spacer M13 ) that is flanked by the 28-nt off-target repeat sequences (repeat NCA ). b , Multiple sequence alignment of the NCA repeats aligned to the BL21 CRISPR repeat sequence. Residues conserved with the BL21 repeat are shown in black. c , Results of the plasmid interference assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing the M13 spacer, or a strain with no plasmid-based array. n = 4 biological replicates (circles). Error bars represent mean ± s.d. c.f.u., colony-forming unit. d , Results of the primed acquisition assay with the strains harbouring the plasmids that encode either WT (BL21) or NCAs containing strains. Black bars correspond to the percentage of total arrays containing new M13-derived spacers. The red bars correspond to the percentage of newly expanded arrays containing M13-derived spacers. n = 3 biological replicates. Error bars represent mean ± s.d. e , Comparison of plasmid-based NCA expansion frequencies following DSA. Expansion frequencies for each NCA were quantified by high-throughput sequencing of the plasmid-based arrays. Each point represents the percentage of expansions detected for each array. We did not detect any expansions for NCAs that do not display a point (fic, potG and yfic) , indicating integration efficiencies of

    Article Snippet: Briefly, liquid cultures of E. coli BL21-AI cells (Thermo) harbouring a plasmid expressing Cas1 and Cas2 under the control of a T7-lac promoter (pWUR 1 + 2, which was a generous gift from U. Qimron) were started from plates and grown overnight in LB.

    Techniques: Activity Assay, Plasmid Preparation, Expressing, Sequencing, CRISPR, Derivative Assay, Next-Generation Sequencing

    TARBP2 is mainly SUMOylated at K52. ( a ) Mutation K52R abolishes TARBP2 SUMOylation in 293T cells. pLPC-TARBP2-WT or -K52R with His-SUMO1/Flag-Ubc9 was co-transfected into 293T cells. Forty-eight hours after transfection, cells were lysed and Ni 2+ -NTA pull down was performed to detect TARBP2 SUMOylation. ( b ) Mutation K52R impairs TARBP2 SUMOylation in an E. coli system. Plasmid pGEX4T1-TARBP2-WT or -K52R with pE1E2S1 plasmid were co-transfected into E. coli BL21 (DE3). Western blot analysis was conducted with anti-SUMO1 antibody after GST pull down, and the same membrane was also detected with anti-TARBP2 antibody after stripping. ( c ) Hypoxia downregulates SUMOylation of TARBP2. 293T cells transfected with indicated plasmids were cultured under 1% of oxygen condition (hypoxia) for indicated time before being harvested. Ni 2+ -NTA resin pull down was performed to detect SUMOylated TARBP2. ( d ) Hydrogen peroxide (H 2 O 2 ) upregulates SUMOylation of TARBP2. 293T cells transfected with indicated plasmids were treated with 100 μM of H 2 O 2 for indicated time before being harvested. Ni 2+ -NTA resin pull down was performed to detect SUMOylated TARBP2. For full scans of western blots ( a – d ), see Supplementary Fig. 8 .

    Journal: Nature Communications

    Article Title: SUMOylation of TARBP2 regulates miRNA/siRNA efficiency

    doi: 10.1038/ncomms9899

    Figure Lengend Snippet: TARBP2 is mainly SUMOylated at K52. ( a ) Mutation K52R abolishes TARBP2 SUMOylation in 293T cells. pLPC-TARBP2-WT or -K52R with His-SUMO1/Flag-Ubc9 was co-transfected into 293T cells. Forty-eight hours after transfection, cells were lysed and Ni 2+ -NTA pull down was performed to detect TARBP2 SUMOylation. ( b ) Mutation K52R impairs TARBP2 SUMOylation in an E. coli system. Plasmid pGEX4T1-TARBP2-WT or -K52R with pE1E2S1 plasmid were co-transfected into E. coli BL21 (DE3). Western blot analysis was conducted with anti-SUMO1 antibody after GST pull down, and the same membrane was also detected with anti-TARBP2 antibody after stripping. ( c ) Hypoxia downregulates SUMOylation of TARBP2. 293T cells transfected with indicated plasmids were cultured under 1% of oxygen condition (hypoxia) for indicated time before being harvested. Ni 2+ -NTA resin pull down was performed to detect SUMOylated TARBP2. ( d ) Hydrogen peroxide (H 2 O 2 ) upregulates SUMOylation of TARBP2. 293T cells transfected with indicated plasmids were treated with 100 μM of H 2 O 2 for indicated time before being harvested. Ni 2+ -NTA resin pull down was performed to detect SUMOylated TARBP2. For full scans of western blots ( a – d ), see Supplementary Fig. 8 .

    Article Snippet: Briefly, E. coli BL21 expressing GEX4T1-TARBP2-WT or -K52R was lysed with B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA).

    Techniques: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Stripping Membranes, Cell Culture

    SUMOylation of TARBP2 increases its binding with Ago2. ( a , b ) The SUMO-site mutation K52R of TARBP2 affects the interaction between TARBP2 and Ago2. 293T cells were transfected Flag-TARBP2-WT or -K52R with ( a ) or without ( b ) myc-Ago2. Forty-eight hours after transfection, cells were lysed for IP with anti-Flag antibody, followed by western blotting with anti-Myc ( a ) or anti-Ago2 ( b ) antibody, respectively. ( c ) SUMOylation enhances TARBP2 binding with Ago2. 293T cells transfected with Flag-TARBP2 were treated with 100 μM H 2 O 2 for 3 h before being harvested. Cell lysates were used for IP with anti-Flag antibody, followed by western blotting with anti-Myc antibody. ( d ) SUMO1 interacts with two SIMs of Ago2. Upper panels: putative SIMs of Ago2 and their mutations (SIM1-wt: 162 L-D-V-V 165 , SIM1-mu: A-A-A-A; SIM-2-wt: 519 V-V-I-L 522 , SIM2-mu: A-A-A-A. Lower panels: plasmid myc-Ago2-WT, -SIM1-mu or -SIM2-mu with/without GFP-SUMO1 were transfected into 293T cells. IP with anti-Myc antibody and immunoblotting with anti-GFP antibody were performed. ( e ) Ago2 interacts with SUMO1 conjugated to TARBP2 via its SIMs. pGEX4T1-TARBP2 with or without pE1E2S1 were co-transfected into E. coli BL21 (DE3). GST-TARBP2 protein was purified, and then immunoblotting with anti-SUMO1 and anti-GST antibodies was performed (left panels). This validated that highly SUMOylated GST-TARBP2 was used for pull down of the same amount of each lysate from 293T cells transfected with the control vector, myc-Ago2 WT or SIM1-mu or SIM2-mu, followed by immunoblotting with anti-Myc antibody (right panels). Cell lysates were also used as an Input. ( f ) The SUMO-site mutation K52R of TARBP2 decreases Ago2. Stable endogenous TARBP2-knocked down 293T cell line (namely TARBP2sh 293 T) was generated by the lentiviral system containing shRNA targeting 3′-UTR of TARBP2 mRNA (left panels). TARBP2sh 293T cells were transfected with Flag-TARBP2-WT or -K52R. Immunoblotting with anti-Dicer, anti-Ago2, anti-Flag and anti-GAPDH antibodies was performed. ( g ) SUMOylation of TARBP2 stabilizes Ago2. Stable HeLa cell lines expressing Flag-TARBP2-WT or -K52R were treated with 100 μg ml −1 CHX for indicated time and cell lysates were immunoblotted with anti-Ago2, anti-Flag and anti-GAPDH antibodies. For full scans of western blots ( a , b , d , g ), see Supplementary Fig. 8 .

    Journal: Nature Communications

    Article Title: SUMOylation of TARBP2 regulates miRNA/siRNA efficiency

    doi: 10.1038/ncomms9899

    Figure Lengend Snippet: SUMOylation of TARBP2 increases its binding with Ago2. ( a , b ) The SUMO-site mutation K52R of TARBP2 affects the interaction between TARBP2 and Ago2. 293T cells were transfected Flag-TARBP2-WT or -K52R with ( a ) or without ( b ) myc-Ago2. Forty-eight hours after transfection, cells were lysed for IP with anti-Flag antibody, followed by western blotting with anti-Myc ( a ) or anti-Ago2 ( b ) antibody, respectively. ( c ) SUMOylation enhances TARBP2 binding with Ago2. 293T cells transfected with Flag-TARBP2 were treated with 100 μM H 2 O 2 for 3 h before being harvested. Cell lysates were used for IP with anti-Flag antibody, followed by western blotting with anti-Myc antibody. ( d ) SUMO1 interacts with two SIMs of Ago2. Upper panels: putative SIMs of Ago2 and their mutations (SIM1-wt: 162 L-D-V-V 165 , SIM1-mu: A-A-A-A; SIM-2-wt: 519 V-V-I-L 522 , SIM2-mu: A-A-A-A. Lower panels: plasmid myc-Ago2-WT, -SIM1-mu or -SIM2-mu with/without GFP-SUMO1 were transfected into 293T cells. IP with anti-Myc antibody and immunoblotting with anti-GFP antibody were performed. ( e ) Ago2 interacts with SUMO1 conjugated to TARBP2 via its SIMs. pGEX4T1-TARBP2 with or without pE1E2S1 were co-transfected into E. coli BL21 (DE3). GST-TARBP2 protein was purified, and then immunoblotting with anti-SUMO1 and anti-GST antibodies was performed (left panels). This validated that highly SUMOylated GST-TARBP2 was used for pull down of the same amount of each lysate from 293T cells transfected with the control vector, myc-Ago2 WT or SIM1-mu or SIM2-mu, followed by immunoblotting with anti-Myc antibody (right panels). Cell lysates were also used as an Input. ( f ) The SUMO-site mutation K52R of TARBP2 decreases Ago2. Stable endogenous TARBP2-knocked down 293T cell line (namely TARBP2sh 293 T) was generated by the lentiviral system containing shRNA targeting 3′-UTR of TARBP2 mRNA (left panels). TARBP2sh 293T cells were transfected with Flag-TARBP2-WT or -K52R. Immunoblotting with anti-Dicer, anti-Ago2, anti-Flag and anti-GAPDH antibodies was performed. ( g ) SUMOylation of TARBP2 stabilizes Ago2. Stable HeLa cell lines expressing Flag-TARBP2-WT or -K52R were treated with 100 μg ml −1 CHX for indicated time and cell lysates were immunoblotted with anti-Ago2, anti-Flag and anti-GAPDH antibodies. For full scans of western blots ( a , b , d , g ), see Supplementary Fig. 8 .

    Article Snippet: Briefly, E. coli BL21 expressing GEX4T1-TARBP2-WT or -K52R was lysed with B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA).

    Techniques: Binding Assay, Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Purification, Generated, shRNA, Expressing

    Effects of recombinant TPY SufS on the growth of E. coli BL21 in the absence and the presence of sodium thiosulfate. a The variation of cell density of E. coli BL21 cells in which recombinant TPY SufS was over-expressed in vivo; b the variations of cell density of E. coli BL21 when directly adding purified TPY SufS (520 ng/mL) and actin protein (520 ng/mL) into medium; c the variation of cell density of E. coli BL21 with over-expression of recombinant TPY SufS in the presence of sodium thiosulfate of 6% (w/v)

    Journal: 3 Biotech

    Article Title: Expression, purification and function of cysteine desulfurase from Sulfobacillus acidophilus TPY isolated from deep-sea hydrothermal vent

    doi: 10.1007/s13205-017-0995-z

    Figure Lengend Snippet: Effects of recombinant TPY SufS on the growth of E. coli BL21 in the absence and the presence of sodium thiosulfate. a The variation of cell density of E. coli BL21 cells in which recombinant TPY SufS was over-expressed in vivo; b the variations of cell density of E. coli BL21 when directly adding purified TPY SufS (520 ng/mL) and actin protein (520 ng/mL) into medium; c the variation of cell density of E. coli BL21 with over-expression of recombinant TPY SufS in the presence of sodium thiosulfate of 6% (w/v)

    Article Snippet: DH5α competent cells and E. coli BL21 competent cells were from Thermo Fisher Scientific (Waltham, MA, USA) and were preserved in −80 °C until use.

    Techniques: Recombinant, In Vivo, Purification, Over Expression

    SDS-PAGE analysis of purified cysteine desulfurase (SufS) over-expressed in E. coli BL21. a Lane1, marker (kDa); lane2, crude extract of supernatant of cells transformed with pET-His; lane3, crude extract of cells transformed with pET-His-SufS; lane4, crude extract of pellets after lysing cells transformed with pET-His-SufS; lane5, crude extract of supernatant after lysing cells transformed with pET-His-SufS. b Lane1, marker (kDa); lane2, purified TPY SufS

    Journal: 3 Biotech

    Article Title: Expression, purification and function of cysteine desulfurase from Sulfobacillus acidophilus TPY isolated from deep-sea hydrothermal vent

    doi: 10.1007/s13205-017-0995-z

    Figure Lengend Snippet: SDS-PAGE analysis of purified cysteine desulfurase (SufS) over-expressed in E. coli BL21. a Lane1, marker (kDa); lane2, crude extract of supernatant of cells transformed with pET-His; lane3, crude extract of cells transformed with pET-His-SufS; lane4, crude extract of pellets after lysing cells transformed with pET-His-SufS; lane5, crude extract of supernatant after lysing cells transformed with pET-His-SufS. b Lane1, marker (kDa); lane2, purified TPY SufS

    Article Snippet: DH5α competent cells and E. coli BL21 competent cells were from Thermo Fisher Scientific (Waltham, MA, USA) and were preserved in −80 °C until use.

    Techniques: SDS Page, Purification, Marker, Transformation Assay, Positron Emission Tomography