protein purification bl21  (Millipore)


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    Protein purification
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    p0181
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    Millipore protein purification bl21

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    Average 99 stars, based on 1 article reviews
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    Clone Assay:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: .. TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. The amplified fragment was cloned into the N-terminal TEV cleavable 6×-His-tag containing vector pNIC28-Bsa4 or the C-terminal TEV cleavable 10×-His-tag vector pNYCOMPS-LIC-TH10-ccdB by ligation-independent cloning.

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: .. Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: .. Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The Eaf1 mutants were constructed using the QuikChange Site-Directed Mutagenesis kit (Strategene).

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: .. Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. Point mutations were carried out with Q5® Site-directed mutagenesis kit (New England BioLabs) following the recommended procedure and done on dsRDB-pET11d vector.

    Amplification:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: .. TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. The amplified fragment was cloned into the N-terminal TEV cleavable 6×-His-tag containing vector pNIC28-Bsa4 or the C-terminal TEV cleavable 10×-His-tag vector pNYCOMPS-LIC-TH10-ccdB by ligation-independent cloning.

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: .. Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Construct:

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The Eaf1 mutants were constructed using the QuikChange Site-Directed Mutagenesis kit (Strategene).

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The verified construct was transformed into E. coli BL21 (DE3) pLysS cells.

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: .. Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. Point mutations were carried out with Q5® Site-directed mutagenesis kit (New England BioLabs) following the recommended procedure and done on dsRDB-pET11d vector.

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: .. Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Nuclear Magnetic Resonance:

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. For NMR experiments, cells containing the plasmid encoding the dsRDB were grown in M9 minimal medium supplemented with 1 g/l of 15 NH4 Cl for 15 N-labeling or 1 g/l 15 NH4 Cl and 2 g/l 13 C-glucose for 13 C/15 N-labeling.

    Infection:

    Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
    Article Snippet: .. Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

    Expressing:

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: .. Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: .. Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The Eaf1 mutants were constructed using the QuikChange Site-Directed Mutagenesis kit (Strategene).

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: .. Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The verified construct was transformed into E. coli BL21 (DE3) pLysS cells.

    Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
    Article Snippet: Protein purification WT Erv1 and cysteine residue mutants were expressed in the E. coli strain Rosetta-gami™ 2 (Novagen). .. Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice.

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    BIA-KA:

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Protein concentration was measured using a BCA kit (Thermofisher).

    Modification:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. HUVEC endothelial cell migration was studied in modified Boyden chambers containing chemotaxis membranes with an 8-µm pore size (Neuroprobe, Gaithersburg, MD) which were coated with Collagen 1 (Cohesion, Palo Alto, CA).

    Western Blot:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. Purification of PEDF protein was determined by Coomassie-stained SDS-polyacrylamide gels and Western blot (results not shown).

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The presence of recombinant protein was confirmed by SDS-PAGE and Western Blot analysis against an anti-His antibody (Novagen, Madison, WI, USA).

    Transformation Assay:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. Vectors containing the cloned target were transformed into Escherichia coli BL21 (DE3) containing the pRIL plasmid (Stratagene) and used to inoculate a 20 mL culture of 2× YT containing 50 μg mL–1 kanamycin and 34 μg mL–1 chloramphenicol.

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The verified construct was transformed into E. coli BL21 (DE3) pLysS cells.

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: .. Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Transfection:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: .. For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. The eluted sample was dialyzed against PBS using a dialysis cassette with 10-kDa cutoff (Pierce Chemical Co, Rockford, IL).

    Chromatography:

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Ligation:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. The amplified fragment was cloned into the N-terminal TEV cleavable 6×-His-tag containing vector pNIC28-Bsa4 or the C-terminal TEV cleavable 10×-His-tag vector pNYCOMPS-LIC-TH10-ccdB by ligation-independent cloning.

    Protease Inhibitor:

    Article Title: Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function
    Article Snippet: Protein purification WT Erv1 and tryptophan mutants were expressed in E. coli strain Rosetta-gami™ 2 (Novagen) and purified as previously described [ , ]. .. Briefly, protein was expressed in the presence of 0.5 mM IPTG and 10 μM FAD at 16 °C for 16–20 h. Induced cell pellets resuspended in buffer A (150 mM NaCl, 50 mM Tris/HCl, pH 7.4) containing 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) were lysed by sonication on ice.

    Article Title: Is a fully wrapped SSB–DNA complex essential for Escherichia coli survival?
    Article Snippet: .. Protein purification wtSSB and the SSB LD proteins were purified as described ( ) with the addition of a double-stranded DNA cellulose column to remove a potential nuclease contaminant ( ) and the inclusion of 1× final concentration of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, Product # S8820) prior to lysing the cells. .. The proteins SSB LD (αΔW), SSB LD (αΔW F) and SSB LD (βΔW) were purified in the same way, except that they were loaded onto the single-stranded DNA cellulose column in [50 mM Tris (pH 8.3), 1 mM Na3 EDTA, 100 mM NaCl and 10% glycerol] instead of the same buffer with 300 mM NaCl used to purify wtSSB and SSB LD.

    Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
    Article Snippet: Protein purification WT Erv1 and cysteine residue mutants were expressed in the E. coli strain Rosetta-gami™ 2 (Novagen). .. Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice.

    Angiogenesis Assay:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: Paragraph title: In Vitro Angiogenesis Assay ... For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany).

    Cell Culture:

    Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
    Article Snippet: Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

    DNA Sequencing:

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Protein Concentration:

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Protein concentration was measured using a BCA kit (Thermofisher).

    Sequencing:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. For those TRAPs cloned into pNIC28-Bsa4, the periplasmic signal sequence, as predicted by SignalP, was not included in the final cloned product.

    Sonication:

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The cells were grown at 37 °C in LB medium containing 0.05 mg/mL ampicillin or kanamycin to log phase and induced with 0.25 mM IPTG at 16 °C for 24 h. The cells were collected and sonicated in a lysis buffer (20 mM Tris-HCl pH 8.0, 1 mM MgCl2 , 150 mM NaCl, and 1 mM PMSF) and then centrifuged at 40,000 × g for 40 min. Eaf1 or Yaf9 was purified separately by affinity chromatography using a GST column (GE Healthcare) or a MBP column, respectively.

    Article Title: Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function
    Article Snippet: Protein purification WT Erv1 and tryptophan mutants were expressed in E. coli strain Rosetta-gami™ 2 (Novagen) and purified as previously described [ , ]. .. Briefly, protein was expressed in the presence of 0.5 mM IPTG and 10 μM FAD at 16 °C for 16–20 h. Induced cell pellets resuspended in buffer A (150 mM NaCl, 50 mM Tris/HCl, pH 7.4) containing 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) were lysed by sonication on ice.

    Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
    Article Snippet: Protein purification WT Erv1 and cysteine residue mutants were expressed in the E. coli strain Rosetta-gami™ 2 (Novagen). .. Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice.

    Recombinant:

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The recombinant proteins were expressed in Escherichia coli BL21 (DE3) Codon-Plus strain (Novagen).

    Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
    Article Snippet: .. Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. Biosynthesis of recombinant protein was induced by adding 0.1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG), and purified by Ni-NTA Superflow resins (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions.

    Mutagenesis:

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The Eaf1 mutants were constructed using the QuikChange Site-Directed Mutagenesis kit (Strategene).

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. Point mutations were carried out with Q5® Site-directed mutagenesis kit (New England BioLabs) following the recommended procedure and done on dsRDB-pET11d vector.

    Purification:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: .. For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. The eluted sample was dialyzed against PBS using a dialysis cassette with 10-kDa cutoff (Pierce Chemical Co, Rockford, IL).

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: .. Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The cells were grown at 37 °C in LB medium containing 0.05 mg/mL ampicillin or kanamycin to log phase and induced with 0.25 mM IPTG at 16 °C for 24 h. The cells were collected and sonicated in a lysis buffer (20 mM Tris-HCl pH 8.0, 1 mM MgCl2 , 150 mM NaCl, and 1 mM PMSF) and then centrifuged at 40,000 × g for 40 min. Eaf1 or Yaf9 was purified separately by affinity chromatography using a GST column (GE Healthcare) or a MBP column, respectively.

    Article Title: Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex *
    Article Snippet: .. Protein Purification MCM complexes, MCM·Cdt1 complexes, ORC complexes, Cdc6, and individual MCM subunits were purified as in Ref. . tc-MCM was prepared as for MCM, except for an additional pulldown step with M2 anti-FLAG resin (Sigma) after purification to deplete endogenous 3×FLAG-tagged wild-type Mcm6. .. Purification of firing factors and execution of reconstituted replication assays in e and d was as in Ref. .

    Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
    Article Snippet: Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. Insect cells were harvested and AtACT2 and AtACT7 were purified as described previously .

    Article Title: Is a fully wrapped SSB–DNA complex essential for Escherichia coli survival?
    Article Snippet: .. Protein purification wtSSB and the SSB LD proteins were purified as described ( ) with the addition of a double-stranded DNA cellulose column to remove a potential nuclease contaminant ( ) and the inclusion of 1× final concentration of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, Product # S8820) prior to lysing the cells. .. The proteins SSB LD (αΔW), SSB LD (αΔW F) and SSB LD (βΔW) were purified in the same way, except that they were loaded onto the single-stranded DNA cellulose column in [50 mM Tris (pH 8.3), 1 mM Na3 EDTA, 100 mM NaCl and 10% glycerol] instead of the same buffer with 300 mM NaCl used to purify wtSSB and SSB LD.

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: .. Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The verified construct was transformed into E. coli BL21 (DE3) pLysS cells.

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: .. Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. Point mutations were carried out with Q5® Site-directed mutagenesis kit (New England BioLabs) following the recommended procedure and done on dsRDB-pET11d vector.

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells
    Article Snippet: .. Protein Purification GST, GST-tagged Igl repeats 19–24 of human FLNa and GST-tagged cytoplasmic domain of CD18 (GST-CD18cyt ) were expressed in BL21 (DE3) cells and purified using glutathione-sepharose (Novagen or GE Healthcare) according to the manufacturer's instructions. .. Purity of these proteins was assessed by SDS-PAGE followed by Commassie staining (Figure ).

    Protein Purification:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: .. For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. The eluted sample was dialyzed against PBS using a dialysis cassette with 10-kDa cutoff (Pierce Chemical Co, Rockford, IL).

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: .. TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. The amplified fragment was cloned into the N-terminal TEV cleavable 6×-His-tag containing vector pNIC28-Bsa4 or the C-terminal TEV cleavable 10×-His-tag vector pNYCOMPS-LIC-TH10-ccdB by ligation-independent cloning.

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: .. Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The Eaf1 mutants were constructed using the QuikChange Site-Directed Mutagenesis kit (Strategene).

    Article Title: Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex *
    Article Snippet: .. Protein Purification MCM complexes, MCM·Cdt1 complexes, ORC complexes, Cdc6, and individual MCM subunits were purified as in Ref. . tc-MCM was prepared as for MCM, except for an additional pulldown step with M2 anti-FLAG resin (Sigma) after purification to deplete endogenous 3×FLAG-tagged wild-type Mcm6. .. Purification of firing factors and execution of reconstituted replication assays in e and d was as in Ref. .

    Article Title: Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells
    Article Snippet: .. Protein purification AtACT2 and AtACT7 were expressed in Sf9 insect cells (Novagen, WI, USA) by infection with recombinant baculoviruses carrying AtACT2-thymosin-His or AtACT2-thymosin-His. .. The infected cells were cultured at 28 °C in 175 cm2 flasks and harvested after 3 days.

    Article Title: Is a fully wrapped SSB–DNA complex essential for Escherichia coli survival?
    Article Snippet: .. Protein purification wtSSB and the SSB LD proteins were purified as described ( ) with the addition of a double-stranded DNA cellulose column to remove a potential nuclease contaminant ( ) and the inclusion of 1× final concentration of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, Product # S8820) prior to lysing the cells. .. The proteins SSB LD (αΔW), SSB LD (αΔW F) and SSB LD (βΔW) were purified in the same way, except that they were loaded onto the single-stranded DNA cellulose column in [50 mM Tris (pH 8.3), 1 mM Na3 EDTA, 100 mM NaCl and 10% glycerol] instead of the same buffer with 300 mM NaCl used to purify wtSSB and SSB LD.

    Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
    Article Snippet: .. Protein purification WT Erv1 and cysteine residue mutants were expressed in the E. coli strain Rosetta-gami™ 2 (Novagen). ..

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: .. Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. Point mutations were carried out with Q5® Site-directed mutagenesis kit (New England BioLabs) following the recommended procedure and done on dsRDB-pET11d vector.

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: .. Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells
    Article Snippet: .. Protein Purification GST, GST-tagged Igl repeats 19–24 of human FLNa and GST-tagged cytoplasmic domain of CD18 (GST-CD18cyt ) were expressed in BL21 (DE3) cells and purified using glutathione-sepharose (Novagen or GE Healthcare) according to the manufacturer's instructions. .. Purity of these proteins was assessed by SDS-PAGE followed by Commassie staining (Figure ).

    Polymerase Chain Reaction:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: .. TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. The amplified fragment was cloned into the N-terminal TEV cleavable 6×-His-tag containing vector pNIC28-Bsa4 or the C-terminal TEV cleavable 10×-His-tag vector pNYCOMPS-LIC-TH10-ccdB by ligation-independent cloning.

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: .. Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Positron Emission Tomography:

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: .. Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The verified construct was transformed into E. coli BL21 (DE3) pLysS cells.

    Lysis:

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The cells were grown at 37 °C in LB medium containing 0.05 mg/mL ampicillin or kanamycin to log phase and induced with 0.25 mM IPTG at 16 °C for 24 h. The cells were collected and sonicated in a lysis buffer (20 mM Tris-HCl pH 8.0, 1 mM MgCl2 , 150 mM NaCl, and 1 mM PMSF) and then centrifuged at 40,000 × g for 40 min. Eaf1 or Yaf9 was purified separately by affinity chromatography using a GST column (GE Healthcare) or a MBP column, respectively.

    SDS Page:

    Article Title: Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis
    Article Snippet: Expression and purification of FvUGT1 protein The cDNA fragment of FvUGT1 was subcloned into Kpn I and Bam HI sites of pET-32 (Novagen, Madison, WI, USA) in frame with both N- and C-terminal His-tags. .. The presence of recombinant protein was confirmed by SDS-PAGE and Western Blot analysis against an anti-His antibody (Novagen, Madison, WI, USA).

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells
    Article Snippet: Protein Purification GST, GST-tagged Igl repeats 19–24 of human FLNa and GST-tagged cytoplasmic domain of CD18 (GST-CD18cyt ) were expressed in BL21 (DE3) cells and purified using glutathione-sepharose (Novagen or GE Healthcare) according to the manufacturer's instructions. .. Purity of these proteins was assessed by SDS-PAGE followed by Commassie staining (Figure ).

    Plasmid Preparation:

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
    Article Snippet: TRAP SBP Cloning and Protein Purification TRAP SBPs were amplified from genomic DNA by PCR using KOD hot start DNA polymerase (Novagen). .. The amplified fragment was cloned into the N-terminal TEV cleavable 6×-His-tag containing vector pNIC28-Bsa4 or the C-terminal TEV cleavable 10×-His-tag vector pNYCOMPS-LIC-TH10-ccdB by ligation-independent cloning.

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The recombinant vector was confirmed with DNA sequencing by the Yale Keck Facility.

    Article Title: Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2
    Article Snippet: .. Protein purification hDus2 or its dsRBD (T339-K451 construct) containing both the NTE and CTE was cloned in a pET11d vector between BamHI and NcoI and expressed in BL21(DE3)pLysS or BL21(DE3) respectively (Novagen) in LB medium and was purified as previously described ( ). .. Point mutations were carried out with Q5® Site-directed mutagenesis kit (New England BioLabs) following the recommended procedure and done on dsRDB-pET11d vector.

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: .. Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Negative Control:

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. Empty pET23b+ was used as a negative control.

    Binding Assay:

    Article Title: Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function
    Article Snippet: Protein purification WT Erv1 and tryptophan mutants were expressed in E. coli strain Rosetta-gami™ 2 (Novagen) and purified as previously described [ , ]. .. Bind resin (Novagen) pre-equilibrated with binding buffer.

    In Vitro:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: Paragraph title: In Vitro Angiogenesis Assay ... For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany).

    Article Title: Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice
    Article Snippet: Translation of rMgt and expression and purification of truncated rMgt protein The coding region of Mgt (GenPept accession no. WP_006806773) from L. goodfellowii was amplified with PCR from genomic DNA from L. goodfellowii with specific primers ( ) and then cloned into EcoR I /Xho I digested pET23b+ (EMD Millipore). .. The in vitro translation of Mgt was performed in an E. coli High-yield Protein Expression System (S30 T7; Promega) according to the manufacturer’s instructions.

    Selection:

    Article Title: Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis
    Article Snippet: .. Plasmid Constructs and Protein Purification WT rv2466c was introduced into pET28B (Novagen), and the resulting plasmid was transformed into BL21(DE3) competent cells (Life technologies) with kanamycin selection (50 μg/mL). .. Bacteria were grown initially at 37 °C while shaking, and protein expression was induced using 0.75 mM IPTG at 16 °C for 18 h. Cells were pelleted and lysed using a sonicator and His tagged protein was purified with nickel chromatography and dialyzed into a 50 mM Tris, pH 7.5, 50 mM NaCl solution.

    Affinity Chromatography:

    Article Title: Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans
    Article Snippet: Bacterial expression and protein purification EAF1 was cloned into pGEX-4T1 with an N-terminal GST tag and YAF9 was cloned into pSJ8 with an N-terminal MBP tag (Novagen). .. The cells were grown at 37 °C in LB medium containing 0.05 mg/mL ampicillin or kanamycin to log phase and induced with 0.25 mM IPTG at 16 °C for 24 h. The cells were collected and sonicated in a lysis buffer (20 mM Tris-HCl pH 8.0, 1 mM MgCl2 , 150 mM NaCl, and 1 mM PMSF) and then centrifuged at 40,000 × g for 40 min. Eaf1 or Yaf9 was purified separately by affinity chromatography using a GST column (GE Healthcare) or a MBP column, respectively.

    Concentration Assay:

    Article Title: Is a fully wrapped SSB–DNA complex essential for Escherichia coli survival?
    Article Snippet: .. Protein purification wtSSB and the SSB LD proteins were purified as described ( ) with the addition of a double-stranded DNA cellulose column to remove a potential nuclease contaminant ( ) and the inclusion of 1× final concentration of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, Product # S8820) prior to lysing the cells. .. The proteins SSB LD (αΔW), SSB LD (αΔW F) and SSB LD (βΔW) were purified in the same way, except that they were loaded onto the single-stranded DNA cellulose column in [50 mM Tris (pH 8.3), 1 mM Na3 EDTA, 100 mM NaCl and 10% glycerol] instead of the same buffer with 300 mM NaCl used to purify wtSSB and SSB LD.

    Chemotaxis Assay:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. HUVEC endothelial cell migration was studied in modified Boyden chambers containing chemotaxis membranes with an 8-µm pore size (Neuroprobe, Gaithersburg, MD) which were coated with Collagen 1 (Cohesion, Palo Alto, CA).

    Migration:

    Article Title: Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment
    Article Snippet: For PEDF protein purification, conditioned media from MatLyLu-PEDF transfected cells was purified on aHisTrap HP column according to the manufacturer's instructions (Novagen, Darmstadt, Germany). .. HUVEC endothelial cell migration was studied in modified Boyden chambers containing chemotaxis membranes with an 8-µm pore size (Neuroprobe, Gaithersburg, MD) which were coated with Collagen 1 (Cohesion, Palo Alto, CA).

    Staining:

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells
    Article Snippet: Protein Purification GST, GST-tagged Igl repeats 19–24 of human FLNa and GST-tagged cytoplasmic domain of CD18 (GST-CD18cyt ) were expressed in BL21 (DE3) cells and purified using glutathione-sepharose (Novagen or GE Healthcare) according to the manufacturer's instructions. .. Purity of these proteins was assessed by SDS-PAGE followed by Commassie staining (Figure ).

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  • 99
    Millipore gst prescission protease
    Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant <t>GST-C-110</t> (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged <t>RBD-87</t> I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.
    Gst Prescission Protease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst prescission protease/product/Millipore
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    93
    Millipore bl21 ∆ luxs
    Construction and identification of luxS mutant strain <t>BL21∆luxS</t>
    Bl21 ∆ Luxs, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 ∆ luxs/product/Millipore
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    bl21 ∆ luxs - by Bioz Stars, 2020-04
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    Image Search Results


    Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant GST-C-110 (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged RBD-87 I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.

    Journal: Cell

    Article Title: Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites

    doi: 10.1016/j.cell.2007.11.048

    Figure Lengend Snippet: Accessible hydrophobic residues in the predicted coiled coil are critical for Rab binding. (A) Helical wheel projection of a coiled coil predicted for GCC185 residues 1579−1606. Residues in registers “a-f” were predicted by the Paircoil program. Residues at positions “a” and “d” lie in the dimer interface. Boxed residues are candidates for binding interactions with Rab GTPases. (B, C) Effect of alanine substitutions on Rab binding. Reactions contained wild type or mutant GST-C-110 (B; 3 μM, C; 2 μM) and 35 S-GTPγS-preloaded GTPases (B; 170 pmol Rab9-His, C; 190 pmol His-Rab6). Data are mean ± SD. (D) Mass determination of untagged RBD-87 I1588A/L1595A by multiple angle static light scattering. The gel filtration elution profile of the protein (black line) and molecular mass (grey line) are shown. Polydispersity of the peak was 1.001.

    Article Snippet: RBD-87 was then separated from GST, GST-Prescission Protease and glutathione beads over a solid support and concentrated (Amicon Ultra, 5,000 MWCO, Millipore).

    Techniques: Binding Assay, Mutagenesis, Filtration

    Identification of a GCC185 Rab-binding domain. (A) Constructs used to map Rab-GCC185 interactions; numbers represent amino acid residues. The GRIP domain and a Rab binding domain (RBD) are shown. At right: summary of binding to Rab6 or Rab9. (B) GCC185 preferentially binds Rab6-GTP via residues upstream of the GRIP domain. Reactions contained 50 pmol His-Rab6 (left) or 1.2 nmol His-Rab6, or Rab9-His (right) using 35 S-GTPγS or 3 H-GDP-preloaded GTPase and either GST-C110 or GST-RBD-87 (2.8 μM). (C) The GRIP domain is not sufficient for Rab binding. GST-C-110, C-110 Y/A , or C-72 (2 μM) was incubated with 35 S-GTPγS-preloaded GTPases (∼500 pmol) (as in B. except Rab1 and Rab9 were untagged and or ArlQ71L-His was used). Untagged Rab9/1 and His-Rab6 1−174 were employed. (D) Rab6 specifically competes with Rab9 for GCC185 binding. GST-C-110:Rab9 complexes (pair of lanes in the center) were incubated for 3 min with ten fold excess competitor. Rab9-His was detected by immunoblot using a monoclonal anti-Rab9 antibody that did not cross react with Rab6 (see pair of lanes at far left). Lower panel, same as upper panel using indicated amounts of competitor Rab9. Data are mean ± SD.

    Journal: Cell

    Article Title: Dual GTPase regulation of the GCC185 Golgin: Communication between adjacent Rab6 and Arl1 binding sites

    doi: 10.1016/j.cell.2007.11.048

    Figure Lengend Snippet: Identification of a GCC185 Rab-binding domain. (A) Constructs used to map Rab-GCC185 interactions; numbers represent amino acid residues. The GRIP domain and a Rab binding domain (RBD) are shown. At right: summary of binding to Rab6 or Rab9. (B) GCC185 preferentially binds Rab6-GTP via residues upstream of the GRIP domain. Reactions contained 50 pmol His-Rab6 (left) or 1.2 nmol His-Rab6, or Rab9-His (right) using 35 S-GTPγS or 3 H-GDP-preloaded GTPase and either GST-C110 or GST-RBD-87 (2.8 μM). (C) The GRIP domain is not sufficient for Rab binding. GST-C-110, C-110 Y/A , or C-72 (2 μM) was incubated with 35 S-GTPγS-preloaded GTPases (∼500 pmol) (as in B. except Rab1 and Rab9 were untagged and or ArlQ71L-His was used). Untagged Rab9/1 and His-Rab6 1−174 were employed. (D) Rab6 specifically competes with Rab9 for GCC185 binding. GST-C-110:Rab9 complexes (pair of lanes in the center) were incubated for 3 min with ten fold excess competitor. Rab9-His was detected by immunoblot using a monoclonal anti-Rab9 antibody that did not cross react with Rab6 (see pair of lanes at far left). Lower panel, same as upper panel using indicated amounts of competitor Rab9. Data are mean ± SD.

    Article Snippet: RBD-87 was then separated from GST, GST-Prescission Protease and glutathione beads over a solid support and concentrated (Amicon Ultra, 5,000 MWCO, Millipore).

    Techniques: Binding Assay, Construct, Incubation

    Construction and identification of luxS mutant strain BL21∆luxS

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Construction and identification of luxS mutant strain BL21∆luxS

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Mutagenesis

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Activity Assay, Mutagenesis

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: SDS Page, Purification, Negative Control