glutathione agarose chromatography  (Millipore)


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    Name:
    Glutathione Agarose
    Description:

    Catalog Number:
    g4510
    Price:
    None
    Applications:
    Affinity chromatography using glutathione-agarose permits rapid, mild, non-denaturing and highly selective purification of proteins containing glutathione binding sequences, such as Glutathione S-Transferase (GST), glutathione peroxidase and glyoxalase I.
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    Structured Review

    Millipore glutathione agarose chromatography
    ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with <t>GST-Raf1-RBD</t> (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P

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    1) Product Images from "Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane"

    Article Title: Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027511

    ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P
    Figure Legend Snippet: ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P

    Techniques Used: Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Expressing, FACS, Construct, Activation Assay, Activity Assay, Generated

    Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 µM, 10 min). Cells were processed as described in material and methods (scale bar 10 µm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 µM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C , D , E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A , then stimulated with LPA (20 µM, 5 min, panel B ), IL-6 (1 ng/ml, 5 min, panel C ) or TPA (100 ng/ml, 5 min, panel D ). Lysates immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure.
    Figure Legend Snippet: Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 µM, 10 min). Cells were processed as described in material and methods (scale bar 10 µm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 µM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C , D , E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A , then stimulated with LPA (20 µM, 5 min, panel B ), IL-6 (1 ng/ml, 5 min, panel C ) or TPA (100 ng/ml, 5 min, panel D ). Lysates immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure.

    Techniques Used:

    Related Articles

    Clone Assay:

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    Article Snippet: The coding sequence of PP2CA was cloned into pGEX-4T1. .. Recombinant GST-PP2CA was produced in Escherichia coli (BL21:DE3) and purified through S-linked glutathione agarose affinity chromatography as recommended by the manufacturer (Sigma G4510).

    Article Title: OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation 1
    Article Snippet: The coding region of OsHAD1 was fused with GST tag by cloning in pGEX4T-1 expression vector (Amersham Biosciences). .. Recombinant OsHAD1 was immobilized on GST beads (Sigma; G4510) according to the manufacturer’s protocol.

    Article Title: Characterization of Chemokine Receptor CXCR2 Interacting Proteins Using a Proteomics Approach to Define the CXCR2 "Chemosynapse"
    Article Snippet: Four clones were selected and 3-ml cultures were grown overnight at 37 °C in LB/ampicillin (1 μ g/ml) and the clones that express the optimal amount of protein were selected. .. The lysate was clarified by centrifugation at 13,000 rpm for 10 min, and the clear supernatant was nutated with preswollen glutathione-agarose beads (Sigma, St. Louis, MO, cat no. G 4510) equilibrated with the lysis buffer for 1 h at 4 °C.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: GST purification Human full-length ORF of Fra-2 was cloned in frame into a pGEX-4T-1 vector, which contains GST to generate a GST-Fra-2 fusion protein. .. Cells were harvested and the fusion protein was purified via affinity binding on GST beads (Sigma-Aldrich, G4510).

    Centrifugation:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
    Article Snippet: .. Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.). ..

    Article Title: Interaction between Polyketide Synthase and Transporter Suggests Coupled Synthesis and Export of Virulence Lipid in M. tuberculosis
    Article Snippet: .. Lysates were cleared by centrifugation at 12,000 g and incubated with Glutathione agarose beads (G 4510, Sigma, St. Louis, Missouri, United States) at 4 °C overnight. ..

    Article Title: Phosphorylation of High-Mobility Group Protein A2 by Nek2 Kinase during the First Meiotic Division in Mouse Spermatocytes
    Article Snippet: .. After centrifugation at 12,000 × g , supernatant fractions were incubated with either glutathione-Sepharose beads (G 4510; Sigma-Aldrich, St. Louis, MO), for GST-Nek2, or Nichel-Agarose beads (QIAGEN, Valencia, CA), for His-HMGA2, for 1 h at 4°C under constant shaking. .. After washes in PBS, GST-fusions were eluted with either 100 mM Tris-HCl, pH 8, 250 mM NaCl containing 10 mM glutathione (G 4251; Sigma-Aldrich), whereas His-HMGA2 was eluted with 500 mM imidazole and dialyzed against PBS overnight.

    Article Title: Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1
    Article Snippet: The soluble fraction was separated from the crude extract by centrifugation at 14,000 rpm for 30 min at 4°C. .. GST-fused proteins were purified on GST beads SIGMA (G4510) following same purification protocol with the exception that the Wash buffer did not contain imidazole.

    Article Title: Characterization of Chemokine Receptor CXCR2 Interacting Proteins Using a Proteomics Approach to Define the CXCR2 "Chemosynapse"
    Article Snippet: .. The lysate was clarified by centrifugation at 13,000 rpm for 10 min, and the clear supernatant was nutated with preswollen glutathione-agarose beads (Sigma, St. Louis, MO, cat no. G 4510) equilibrated with the lysis buffer for 1 h at 4 °C. .. The beads with bound GST-fusion proteins were washed thrice with the lysis buffer and once without any detergent in the lysis buffer.

    Construct:

    Article Title: A novel regulatory mechanism links PLC?1 to PDK1
    Article Snippet: All GST constructs were kindly provided by Dario Alessi. .. 500 µg of HEK293 overexpressing GST–empty-vector and 500 µg of HEK293 lysate overexpressing GST–PDK1 were incubated separately with 20 µl of G4510 glutathione–Sepharose beads (Sigma-Aldrich) for 1 hour at 4°C.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: Paragraph title: ELISA ... The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation.

    Incubation:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
    Article Snippet: .. Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.). ..

    Article Title: A novel regulatory mechanism links PLC?1 to PDK1
    Article Snippet: .. 500 µg of HEK293 overexpressing GST–empty-vector and 500 µg of HEK293 lysate overexpressing GST–PDK1 were incubated separately with 20 µl of G4510 glutathione–Sepharose beads (Sigma-Aldrich) for 1 hour at 4°C. .. Beads were washed 3× in washing buffer for 5 minutes at 4°C and incubated with 500 µg of protein lysates from HEK293 overexpressing PLCγ1, overnight on a rotating wheel at 4°C in a total volume of 1 ml.

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: .. The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation. .. After several washes in PBS, protein was eluted in elution buffer (100 mM Tris-HCl, pH 8.0, 10 mM GSH (Sigma, G4251), 300 mM NaCl, 1 mM DTT and protease inhibitors).

    Article Title: Interaction between Polyketide Synthase and Transporter Suggests Coupled Synthesis and Export of Virulence Lipid in M. tuberculosis
    Article Snippet: .. Lysates were cleared by centrifugation at 12,000 g and incubated with Glutathione agarose beads (G 4510, Sigma, St. Louis, Missouri, United States) at 4 °C overnight. ..

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    Article Title: CDKN1C Negatively Regulates RNA Polymerase II C-terminal Domain Phosphorylation in an E2F1-dependent Manner *
    Article Snippet: Beads were washed three times with the same protein lysis buffer, incubated with 800 μg of protein extract for 2 h at 4 °C, and washed again. .. IP and GST bead (G4510, Sigma) pull-down reactions on bacterially expressed proteins were carried out using 20 μg of crude bacterial lysates that were prepared as described above.

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    Expressing:

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    Article Title: OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation 1
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    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
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    Western Blot:

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    Transformation Assay:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
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    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: ELISA Escherichia coli cells (BL21DE3) transformed with pGeX4T1-mutp53R175H vector were grown at 37 °C in LB medium containing 100 μ g/ml ampicillin to an optical density at 600 nm of 0.4. .. The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation.

    Article Title: Phosphorylation of High-Mobility Group Protein A2 by Nek2 Kinase during the First Meiotic Division in Mouse Spermatocytes
    Article Snippet: Escherichia coli cells (BL21-DE3) were transformed with the appropriate plasmid, grown at 37°C in LB medium to an optical density (600 nm) of 0.4, and induced with 0.5 mM isopropyl-1-thio-β-galactopyranoside for 3 h at the same temperature. .. After centrifugation at 12,000 × g , supernatant fractions were incubated with either glutathione-Sepharose beads (G 4510; Sigma-Aldrich, St. Louis, MO), for GST-Nek2, or Nichel-Agarose beads (QIAGEN, Valencia, CA), for His-HMGA2, for 1 h at 4°C under constant shaking.

    Article Title: OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation 1
    Article Snippet: Recombinant OsHAD1 and empty pGEX4T-1 vector were independently transformed and induced in E. coli strain BL21(DE3)pLysS as described above. .. Recombinant OsHAD1 was immobilized on GST beads (Sigma; G4510) according to the manufacturer’s protocol.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: GST-Fra-2 was transformed in BL-21-competent Escherichia coli cells. .. Cells were harvested and the fusion protein was purified via affinity binding on GST beads (Sigma-Aldrich, G4510).

    Transfection:

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    Protease Inhibitor:

    Article Title: Characterization of Chemokine Receptor CXCR2 Interacting Proteins Using a Proteomics Approach to Define the CXCR2 "Chemosynapse"
    Article Snippet: The bacterial pellet was resuspended in PBS with 0.1% Triton and bacterial protease inhibitor cocktail (AEBSF, Bestatin HCl, E-64, EDTA [ethylenediaminetetraacetic acid] and pepstatin A; Sigma, St. Louis, MO, cat. no. P 8465). .. The lysate was clarified by centrifugation at 13,000 rpm for 10 min, and the clear supernatant was nutated with preswollen glutathione-agarose beads (Sigma, St. Louis, MO, cat no. G 4510) equilibrated with the lysis buffer for 1 h at 4 °C.

    Generated:

    Article Title: PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility
    Article Snippet: Rabbit polyclonal antibodies (7801/7802) were generated to mouse p190RhoGEF residues 1457–1582 expressed as a GST fusion pro tein (University of California, San Diego). .. Antibodies were affinity purified using GST-p190RhoGEF (1457–1582) covalently coupled to glutathione agarose (Sigma-Aldrich) using dimethylpimelimidate as described previously ( ).

    other:

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    Protein Concentration:

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    Article Snippet: Determine protein concentration and adjust protein concentration to 1mg/ml with lysis buffer. .. Glutathione agarose should be prepared in advance by swelling glutathione immobilized on cross-linked 4% beaded agarose (Sigma, G-4510) in water at 200 ml/g for 2 hours.

    Sequencing:

    Article Title: ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis [C] [C] [W]
    Article Snippet: The coding sequence of PP2CA was cloned into pGEX-4T1. .. Recombinant GST-PP2CA was produced in Escherichia coli (BL21:DE3) and purified through S-linked glutathione agarose affinity chromatography as recommended by the manufacturer (Sigma G4510).

    Sonication:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
    Article Snippet: Cultures were then diluted 1:20 in fresh medium, grown to an OD600 of 0.7 at 30°C and 200 rpm, and induced by the addition of isopropyl β-d -thiogalactopyranoside (IPTG) to 0.4 mM for an additional 3 h. Bacteria were harvested by centrifugation at 4,000 g , resuspended in PBS containing 1 mM EDTA and 1% (vol/vol) Triton X-100, and lysed by sonication. .. Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.).

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: The pellet was resuspended in sarcosyl buffer (lysis buffer+2% sarcosyl) followed by probe sonication (three cycles of 1 min each). .. The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation.

    Article Title: Interaction between Polyketide Synthase and Transporter Suggests Coupled Synthesis and Export of Virulence Lipid in M. tuberculosis
    Article Snippet: Cells were centrifuged and resuspended in 50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1 mM DTT, and lysed by sonication. .. Lysates were cleared by centrifugation at 12,000 g and incubated with Glutathione agarose beads (G 4510, Sigma, St. Louis, Missouri, United States) at 4 °C overnight.

    Article Title: Phosphorylation of High-Mobility Group Protein A2 by Nek2 Kinase during the First Meiotic Division in Mouse Spermatocytes
    Article Snippet: Cells were harvested by centrifugation and lysed in ice-cold phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 1 mM dithiothreitol (DTT), protease inhibitors, by probe sonication (3 cycles of 1 min). .. After centrifugation at 12,000 × g , supernatant fractions were incubated with either glutathione-Sepharose beads (G 4510; Sigma-Aldrich, St. Louis, MO), for GST-Nek2, or Nichel-Agarose beads (QIAGEN, Valencia, CA), for His-HMGA2, for 1 h at 4°C under constant shaking.

    Affinity Purification:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
    Article Snippet: Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.). .. Recombinant MBP-plectin ABD fusion protein was expressed and purified as described above, except that amylose resin (800-21; New England Biolabs) was used for the affinity purification, and that equilibration and elution of the resin was in 20 mM Tris-HCl (pH 7.4), 1 mM β-mercaptoethanol without or with 10 mM maltose, respectively.

    Article Title: Induction of Glutathione S-Transferases in Arabidopsis by Herbicide Safeners 1
    Article Snippet: The resulting supernatant was applied to GSH-agarose (G-4510, Sigma, St. Louis) packed in a 10-mL column equilibrated with buffer A (20 m m Tris-HCl [pH 7.8], 1 m m EDTA, and 5 m m DTT). .. The affinity-purified GSTs were immediately concentrated and desalted in buffer A using a Centricon YM-10 spin column (10-kD cutoff, Millipore Corp., Bedford, MA) before storage at −70°C.

    Article Title: PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility
    Article Snippet: .. Antibodies were affinity purified using GST-p190RhoGEF (1457–1582) covalently coupled to glutathione agarose (Sigma-Aldrich) using dimethylpimelimidate as described previously ( ). .. Antibody specificity was confirmed by Western blot analysis combined with soluble GST-p190RhoGEF (1457–1582) as a competitive inhibitor. pCDNA3-Y1003A p190RhoGEF was provided by J. Lee (Ewha Womans University, Seoul, Korea).

    Binding Assay:

    Article Title: Interaction between Polyketide Synthase and Transporter Suggests Coupled Synthesis and Export of Virulence Lipid in M. tuberculosis
    Article Snippet: Paragraph title: GST pulldown binding assays. ... Lysates were cleared by centrifugation at 12,000 g and incubated with Glutathione agarose beads (G 4510, Sigma, St. Louis, Missouri, United States) at 4 °C overnight.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. Cells were harvested and the fusion protein was purified via affinity binding on GST beads (Sigma-Aldrich, G4510). .. In vitro kinase assay Bacterially expressed-purified GST (2 μ g), GST-Fra-2 (2 μ g) or MBP (NEB, P6021S; 2 μ g) were incubated with radioactive γ32 P-ATP (Amersham, GE Healthcare) with or without purified ERK 2 (20 ng) kinase (NEB, P6080S).

    Affinity Precipitation:

    Article Title: Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion
    Article Snippet: Paragraph title: Affinity precipitation and detection of activated Ras ... Glutathione agarose should be prepared in advance by swelling glutathione immobilized on cross-linked 4% beaded agarose (Sigma, G-4510) in water at 200 ml/g for 2 hours.

    Pull Down Assay:

    Article Title: A novel regulatory mechanism links PLC?1 to PDK1
    Article Snippet: Paragraph title: GST pull-down assay ... 500 µg of HEK293 overexpressing GST–empty-vector and 500 µg of HEK293 lysate overexpressing GST–PDK1 were incubated separately with 20 µl of G4510 glutathione–Sepharose beads (Sigma-Aldrich) for 1 hour at 4°C.

    Purification:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
    Article Snippet: Paragraph title: Purification of Recombinant Fusion Proteins ... Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.).

    Article Title: Phosphorylation of High-Mobility Group Protein A2 by Nek2 Kinase during the First Meiotic Division in Mouse Spermatocytes
    Article Snippet: Paragraph title: Bacterial Protein Expression and Purification ... After centrifugation at 12,000 × g , supernatant fractions were incubated with either glutathione-Sepharose beads (G 4510; Sigma-Aldrich, St. Louis, MO), for GST-Nek2, or Nichel-Agarose beads (QIAGEN, Valencia, CA), for His-HMGA2, for 1 h at 4°C under constant shaking.

    Article Title: ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis [C] [C] [W]
    Article Snippet: .. Recombinant GST-PP2CA was produced in Escherichia coli (BL21:DE3) and purified through S-linked glutathione agarose affinity chromatography as recommended by the manufacturer (Sigma G4510). .. N- (residues 1 to 293, CD) and C-terminal (residues 294–512, RD) SnRK1.1 were cloned into pET28a (Novagen).

    Article Title: Induction of Glutathione S-Transferases in Arabidopsis by Herbicide Safeners 1
    Article Snippet: Paragraph title: Protein Extraction and Purification ... The resulting supernatant was applied to GSH-agarose (G-4510, Sigma, St. Louis) packed in a 10-mL column equilibrated with buffer A (20 m m Tris-HCl [pH 7.8], 1 m m EDTA, and 5 m m DTT).

    Article Title: PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility
    Article Snippet: Antibodies were affinity purified using GST-p190RhoGEF (1457–1582) covalently coupled to glutathione agarose (Sigma-Aldrich) using dimethylpimelimidate as described previously ( ). .. Purified bovine plasma FN was obtained from EMD.

    Article Title: Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1
    Article Snippet: .. GST-fused proteins were purified on GST beads SIGMA (G4510) following same purification protocol with the exception that the Wash buffer did not contain imidazole. ..

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: .. Cells were harvested and the fusion protein was purified via affinity binding on GST beads (Sigma-Aldrich, G4510). .. In vitro kinase assay Bacterially expressed-purified GST (2 μ g), GST-Fra-2 (2 μ g) or MBP (NEB, P6021S; 2 μ g) were incubated with radioactive γ32 P-ATP (Amersham, GE Healthcare) with or without purified ERK 2 (20 ng) kinase (NEB, P6080S).

    Protein Extraction:

    Article Title: Induction of Glutathione S-Transferases in Arabidopsis by Herbicide Safeners 1
    Article Snippet: Paragraph title: Protein Extraction and Purification ... The resulting supernatant was applied to GSH-agarose (G-4510, Sigma, St. Louis) packed in a 10-mL column equilibrated with buffer A (20 m m Tris-HCl [pH 7.8], 1 m m EDTA, and 5 m m DTT).

    Affinity Chromatography:

    Article Title: ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis [C] [C] [W]
    Article Snippet: .. Recombinant GST-PP2CA was produced in Escherichia coli (BL21:DE3) and purified through S-linked glutathione agarose affinity chromatography as recommended by the manufacturer (Sigma G4510). .. N- (residues 1 to 293, CD) and C-terminal (residues 294–512, RD) SnRK1.1 were cloned into pET28a (Novagen).

    Lysis:

    Article Title: Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion
    Article Snippet: Determine protein concentration and adjust protein concentration to 1mg/ml with lysis buffer. .. Glutathione agarose should be prepared in advance by swelling glutathione immobilized on cross-linked 4% beaded agarose (Sigma, G-4510) in water at 200 ml/g for 2 hours.

    Article Title: A novel regulatory mechanism links PLC?1 to PDK1
    Article Snippet: Twenty-four hours after transfection, cells were lysed in lysis buffer (50 mM Tris pH 8.0, 50 mM KCl, 1% NP-40) containing protease and phosphatases inhibitors (Sigma-Aldrich, UK). .. 500 µg of HEK293 overexpressing GST–empty-vector and 500 µg of HEK293 lysate overexpressing GST–PDK1 were incubated separately with 20 µl of G4510 glutathione–Sepharose beads (Sigma-Aldrich) for 1 hour at 4°C.

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: The pellet was resuspended in sarcosyl buffer (lysis buffer+2% sarcosyl) followed by probe sonication (three cycles of 1 min each). .. The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation.

    Article Title: CDKN1C Negatively Regulates RNA Polymerase II C-terminal Domain Phosphorylation in an E2F1-dependent Manner *
    Article Snippet: Beads were washed three times with the same protein lysis buffer, incubated with 800 μg of protein extract for 2 h at 4 °C, and washed again. .. IP and GST bead (G4510, Sigma) pull-down reactions on bacterially expressed proteins were carried out using 20 μg of crude bacterial lysates that were prepared as described above.

    Article Title: Characterization of Chemokine Receptor CXCR2 Interacting Proteins Using a Proteomics Approach to Define the CXCR2 "Chemosynapse"
    Article Snippet: .. The lysate was clarified by centrifugation at 13,000 rpm for 10 min, and the clear supernatant was nutated with preswollen glutathione-agarose beads (Sigma, St. Louis, MO, cat no. G 4510) equilibrated with the lysis buffer for 1 h at 4 °C. .. The beads with bound GST-fusion proteins were washed thrice with the lysis buffer and once without any detergent in the lysis buffer.

    SDS Page:

    Article Title: Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1
    Article Snippet: Eluted samples were analyzed by SDS PAGE and stained with Coomassie blue. .. GST-fused proteins were purified on GST beads SIGMA (G4510) following same purification protocol with the exception that the Wash buffer did not contain imidazole.

    Plasmid Preparation:

    Article Title: A novel regulatory mechanism links PLC?1 to PDK1
    Article Snippet: Three sets of three 6-cm Petri dishes of HEK293 were transfected with PRK5-PLCγ1, pEBG2T-MYC-PDK1, pEBG2T-MYC-PDK1-L155E, pEBG2T-MYC-PDK1-D223A (PDK1 Kinase-Dead), pEBG2T-MYC-PDK1-(2–450) (PDK1-ΔPH-PDK1), pEBG2T-PDK1 (408–end) (PH-PDK1) and pEBG2T-GST empty vector, respectively. .. 500 µg of HEK293 overexpressing GST–empty-vector and 500 µg of HEK293 lysate overexpressing GST–PDK1 were incubated separately with 20 µl of G4510 glutathione–Sepharose beads (Sigma-Aldrich) for 1 hour at 4°C.

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: ELISA Escherichia coli cells (BL21DE3) transformed with pGeX4T1-mutp53R175H vector were grown at 37 °C in LB medium containing 100 μ g/ml ampicillin to an optical density at 600 nm of 0.4. .. The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation.

    Article Title: Phosphorylation of High-Mobility Group Protein A2 by Nek2 Kinase during the First Meiotic Division in Mouse Spermatocytes
    Article Snippet: Escherichia coli cells (BL21-DE3) were transformed with the appropriate plasmid, grown at 37°C in LB medium to an optical density (600 nm) of 0.4, and induced with 0.5 mM isopropyl-1-thio-β-galactopyranoside for 3 h at the same temperature. .. After centrifugation at 12,000 × g , supernatant fractions were incubated with either glutathione-Sepharose beads (G 4510; Sigma-Aldrich, St. Louis, MO), for GST-Nek2, or Nichel-Agarose beads (QIAGEN, Valencia, CA), for His-HMGA2, for 1 h at 4°C under constant shaking.

    Article Title: OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation 1
    Article Snippet: Recombinant OsHAD1 and empty pGEX4T-1 vector were independently transformed and induced in E. coli strain BL21(DE3)pLysS as described above. .. Recombinant OsHAD1 was immobilized on GST beads (Sigma; G4510) according to the manufacturer’s protocol.

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells
    Article Snippet: GST purification Human full-length ORF of Fra-2 was cloned in frame into a pGEX-4T-1 vector, which contains GST to generate a GST-Fra-2 fusion protein. .. Cells were harvested and the fusion protein was purified via affinity binding on GST beads (Sigma-Aldrich, G4510).

    Recombinant:

    Article Title: Binding of Integrin ?6?4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding
    Article Snippet: Paragraph title: Purification of Recombinant Fusion Proteins ... Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads (G 4510; Sigma Chemical Co.).

    Article Title: Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
    Article Snippet: The expression of recombinant protein was induced by the addition of 0.5 mM isopropyl-1-thio-β -galactopyranoside for 3 h at the same temperature under vigorous shaking. .. The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads (Sigma, St. Louis, MO, USA, G 4510) for 2 h at 4 °C with constant rotation.

    Article Title: Interaction between Polyketide Synthase and Transporter Suggests Coupled Synthesis and Export of Virulence Lipid in M. tuberculosis
    Article Snippet: Recombinant MmpL7 domain 2–GST, MmpL7 domain 2–I611S–GST, and GST alone were expressed in E. coli strain DH5α by growing cultures to mid-logarithmic phase at 32 °C and inducing with 1 mM IPTG for 30 min. .. Lysates were cleared by centrifugation at 12,000 g and incubated with Glutathione agarose beads (G 4510, Sigma, St. Louis, Missouri, United States) at 4 °C overnight.

    Article Title: ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis [C] [C] [W]
    Article Snippet: .. Recombinant GST-PP2CA was produced in Escherichia coli (BL21:DE3) and purified through S-linked glutathione agarose affinity chromatography as recommended by the manufacturer (Sigma G4510). .. N- (residues 1 to 293, CD) and C-terminal (residues 294–512, RD) SnRK1.1 were cloned into pET28a (Novagen).

    Article Title: OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation 1
    Article Snippet: .. Recombinant OsHAD1 was immobilized on GST beads (Sigma; G4510) according to the manufacturer’s protocol. ..

    Produced:

    Article Title: ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis [C] [C] [W]
    Article Snippet: .. Recombinant GST-PP2CA was produced in Escherichia coli (BL21:DE3) and purified through S-linked glutathione agarose affinity chromatography as recommended by the manufacturer (Sigma G4510). .. N- (residues 1 to 293, CD) and C-terminal (residues 294–512, RD) SnRK1.1 were cloned into pET28a (Novagen).

    Staining:

    Article Title: Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1
    Article Snippet: Eluted samples were analyzed by SDS PAGE and stained with Coomassie blue. .. GST-fused proteins were purified on GST beads SIGMA (G4510) following same purification protocol with the exception that the Wash buffer did not contain imidazole.

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