Gst sepharose 4b

Manufactured by GE Healthcare

GST Sepharose 4B is a pre-packed affinity chromatography resin designed for the purification of glutathione S-transferase (GST) fusion proteins. The resin consists of glutathione, which is covalently coupled to Sepharose 4B, a highly cross-linked agarose bead matrix. The GST tag binds to the immobilized glutathione, allowing the target protein to be captured and purified from complex mixtures.

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Lab products found in correlation

Hiload superdex 200 pg column, by GE Healthcare (1 mentions) Bl21 star de3 cells, by Thermo Fisher Scientific (1 mentions) Pgex 4t1 vector, by GenScript (1 mentions) Complete edta free protease inhibitor cocktail, by Roche (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions)

Spelling variants of this product

Sepharose 4B (96 citations) Glutathione Sepharose 4B GST-tagged protein purification resin (13 citations) GST-Sepharose (10 citations) GST-Sepharose 4B resin (5 citations) GST-Sepharose 4B beads (5 citations)

5 protocols using gst sepharose 4b

SARS-CoV-2 Main Protease Purification

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The SARS-CoV2 M^pro-encoding sequence was cloned into pGEX-4T1 vector (Genscript, Piscataway, NJ). The plasmid construct was transformed into BL21 Star (DE3) cells (Thermo Fisher Scientific). The culture was grown in Terrific Broth media supplemented with ampicillin. Protein expression was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside at an optical density at 600 nm of 0.5. Protein expression continued at 20°C overnight. SARS-CoV-2 M^pro was purified first by affinity chromatography using glutathione S-transferase (GST) Sepharose 4B (GE Healthcare, Piscataway, NJ). The GST tag was cleaved off by the use of thrombin and separated from M^pro via GST affinity chromatography, providing the intact M^pro with an additional N-terminal glycine residue. The cleaved M^pro was further purified by size exclusion chromatography using a HiLoad Superdex 200-pg column (GE Healthcare) in a reaction mixture containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 2 mM dithiothreitol. The protease was confirmed to be >99% pure based on SDS-gel electrophoresis and HPLC/MS chromatography as shown in Fig. S7 in the supplemental material.

Hattori S.I., Higshi-Kuwata N., Raghavaiah J., Das D., Bulut H., Davis D.A., Takamatsu Y., Matsuda K., Takamune N., Kishimoto N., Okamura T., Misumi S., Yarchoan R., Maeda K., Ghosh A.K, & Mitsuya H. (2020). GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection. mBio, 11(4), e01833-20.

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Affinity-based Purification and Binding Assays of Recombinant Proteins

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Recombinant proteins were expressed in the BL21 E. coli strain. GST-tagged proteins were then purified with GST Sepharose 4B (GE Life Sciences) and His-tagged proteins were purified with His-Bind Resin (Novagen). Recombinant PI(3)K (p110a and p85α) proteins were purchased from Echelon Biosciences. GST-tagged proteins were incubated with glutathione beads before binding assays. The binding assay was performed in the lysis buffer used for immunoprecipitation by adding 10 nM to 5 µM of His-tagged proteins and 20 µl of GST-tagged protein-bound glutathione beads. After incubation for 1 h at 25 °C, unbound proteins were washed out and the protein complex was analysed by immunoblotting. For the triple binding assay with recombinant GST-IQGAP1, His-PIPKIα and PI(3)K (His-p110oc and untagged p85oc), PIPKIα was pulled down with an anti-PIPKIα antibody pre-bound on Protein G Sepharose beads. For binding assays with p85α, wild-type or deletion mutants of bovine p85α²⁸ were expressed in HEK293 cells and cell lysates were used for binding experiments with recombinant IQGAP1 proteins.

Choi S., Hedman A.C., Sayedyahossein S., Thapa N., Sacks D.B, & Anderson R.A. (2016). Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases. Nature cell biology, 18(12), 1324-1335.

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Recombinant Protein Pulldown Assay

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Recombinant GST-fused proteins in a slurry of GST Sepharose 4B (GE Healthcare) were incubated with the indicated recombinant proteins in binding buffer (10 mM HEPES, pH 7.9, 2.5 mM MgCl₂, 50 mM KCl, 150 mM NaCl, 5% glycerol, 1 mM DTT, and 0.1% NP-40) with rotation for 2 hours at 4°C, followed by pulldown with GST-sepharose. After washing 3 times with the binding buffer, proteins were eluted with 2× SDS sample buffer and 100 mM DTT, followed by SDS-PAGE and immunoblotting.

Kashihara T., Mukai R., Oka S.I., Zhai P., Nakada Y., Yang Z., Mizushima W., Nakahara T., Warren J.S., Abdellatif M, & Sadoshima J. (2022). YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload. The Journal of Clinical Investigation, 132(6), e150595.

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Ubiquitin Enrichment and Western Blot

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Cells were lysed on ice in lysis buffer [200 mM Tris HCL pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% Glycerol, 50 mM NaF, 200 μM NaVO₄, complete protease inhibitor cocktail EDTA-free from Roche, supplemented with fresh PR619 (DUB inhibitor, 1:2000, SI9619 LifeSensors). Supernatant was collected after 10 min centrifugation in a cooling centrifuge. Protein lysate was incubated together with pre-washed glutathione sepharose beads (GST Sepharose 4B, GE Healthcare) and recombinant ubiquitin in a rocker overnight at 4 °C. The next day, the samples were washed five times with washing buffer (PBS, 1% Triton, 1 mM EDTA plus PR619) and centrifuged each time at 4000 rpm for 30 s at 4 °C. After complete removal of the last wash by aspiration, samples were boiled using laemmli (plus DTT) and subjected to SDS-PAGE and Western blot analysis. Primary antibodies used were; ER (Santa-Cruz sc8002) and ubiquitin (P4D1, CST-3936). Secondary antibodies were used at concentration of 1/2000 (Dako, Denmark A/S).

Simigdala N., Pancholi S., Ribas R., Folkerd E., Liccardi G., Nikitorowicz-Buniak J., Johnston S.R., Dowsett M, & Martin L.A. (2018). Abiraterone shows alternate activity in models of endocrine resistant and sensitive disease. British Journal of Cancer, 119(3), 313-322.

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Ubiquitin Interaction Proteomics Protocol

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Cells were lysed on ice in lysis buffer [200 mM Tris HCL pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% Glycerol, 50 mM NaF, 200 μM NaVO4, complete protease inhibitor cocktail EDTA-free from Roche, supplemented with fresh PR619 (DUB inhibitor, 1:2000, SI9619 LifeSensors). Supernatant was collected after 10 minutes centrifugation in a cooling centrifuge. Protein lysate was incubated together with pre-washed glutathione sepharose beads (GST Sepharose 4B, GE Healthcare) and recombinant ubiquitin in a rocker overnight at 4°C. The next day, the samples were washed five times with washing buffer (PBS, 1% Triton, 1 mM EDTA plus PR619) and centrifuged each time at 4000 rpm for 30 sec at 4°C. After complete removal of the last wash by aspiration, samples were boiled using laemmli (plus DTT) and subjected to SDS-PAGE and western blot analysis. Primary antibodies used were; ER (Santa-Cruz sc8002) and ubiquitin (P4D1, CST-3936). Secondary antibodies were used at concentration of 1/2000 (Dako, Denmark A/S).

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