Glutathione sepharose

Manufactured by Thermo Fisher Scientific

Sourced in United States, Ireland

Glutathione-Sepharose is a chromatography resin used for the purification of proteins that have a glutathione S-transferase (GST) tag. It consists of glutathione, a tripeptide, covalently linked to Sepharose beads, which serve as the solid support. The glutathione moiety on the resin binds to the GST tag on the target protein, allowing for its capture and separation from other cellular components during the purification process.

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Lab products found in correlation

Ni nta agarose, by Qiagen (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bradford s reagent, by Bio-Rad (1 mentions) Imidazole, by Thermo Fisher Scientific (1 mentions) Bovine fetuin, by Merck Group (1 mentions) Simple blue stain, by Thermo Fisher Scientific (1 mentions) Citric acid, by Thermo Fisher Scientific (1 mentions) Valproic acid, by Merck Group (1 mentions) Cacl2, by Thermo Fisher Scientific (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions) Hrp linked peanut agglutinin, by Merck Group (1 mentions) O phenylenediamine dihydrochloride, by Merck Group (1 mentions) Freestyle 293 expression media, by Thermo Fisher Scientific (1 mentions) Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Glutathione Sepharose beads (26 citations) Glutathione Sepharose 4B beads (13 citations) Glutathione Sepharose 4B (12 citations) Glutathione-Sepharose 4B resin (2 citations)

8 protocols using glutathione sepharose

GST-RXRα and His-GSK-3β Protein Interaction

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The constructs of pGEX-4T-1-GST-RXRα and pET-22b-His-GSK-3β were transformed into E. coli. BL21 and E. coli. BL21 (DE3) respectively. Both cells were induced with 1 mM IPTG at 25ºC for 16 h. Bacterial pellets were sonicated and centrifuged. The supernatants were mixed with Glutathione-Sepharose (Thermo Scientific) and Ni-NTA agarose (Qiagen), for purification of GST-RXRα and His-GSK-3β, respectively. Immobilized GST-RXRα protein was incubated with 1 μg His-GSK-3β protein in 500 μl of 150 mM NaCl, 100 mM NaF, 50 mM Tris-HCl, pH 7.6, 0.5% NP-40 and 1 mM PMSF at 4 °C for 2 h. After thoroughly washing, bound proteins were eluted with elution buffer (20 mM Tris-HCl, pH 8.0; 10 mM GSH; 1 mM PMSF; 5 mM DTT) and processed for SDS-PAGE.

Zhang S., Gao W., Tang J., Zhang H., Zhou Y., Liu J., Chen K., Liu F., Li W., To S.K., Wong A.S., Zhang X.K., Zhou H, & Zeng J.Z. (2020). The Roles of GSK-3β in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma. Theranostics, 10(3), 1230-1244.

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GST Pull-down Assay for Nur77-Akt Interaction

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GST pull-down assays were carried out as previously described^{39 (link)}. GST-Nur77 LBD or its point mutants, and His-Akt were expressed in Escherichia coli strain BL21, and purified using glutathione Sepharose (Thermo Fisher) or Ni-NTA agarose (Qiagen), respectively. The bead-bound GST-fusion proteins (2 μg) were incubated with His-tagged protein (1 μg) in the presence or absence of PDNPA (10 μM) in 1 ml of modified ELB (50 mM Tris-HCl, pH 7.6, 100 mM NaF, 150 mM NaCl, 0.5% Nonidet P-40 and 1 mM PMSF) at 4 °C for 1 h. Beads were washed with modified ELB three times, and resuspended in SDS loading buffer for western blotting.

Chen Q.T., Zhang Z.Y., Huang Q.L., Chen H.Z., Hong W.B., Lin T., Zhao W.X., Wang X.M., Ju C.Y., Wu L.Z., Huang Y.Y., Hou P.P., Wang W.J., Zhou D., Deng X, & Wu Q. (2022). HK1 from hepatic stellate cell–derived extracellular vesicles promotes progression of hepatocellular carcinoma. Nature Metabolism, 4(10), 1306-1321.

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Expression and Purification of GST-JAK1 NLS

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Recombinant human GST-JAK1 NLS fragment was expressed and purified from bacteria. Briefly, transformed E. coli Rosseta BL21 were grown with vigorous shaking at 37 °C in LB medium. At mid-log phase of growth, protein expression was induced by 0.1 mM isopropyl β–D-1-thiogalactopyranoside (IPTG) for 4 h at 37 °C. The cells were collected and lysed in lysis buffer (1× PBS, 10mM EGTA, 10mM EDTA, 0.1% Tween 20, 250mM NaCl, add DTT, lysozyme and PIC before use). The slurry was sonicated at 40% amplitude: 10 sec on, 20sec off with a total “on” time of 3 mins. The lysate was clarified (15 mins, 15000 rpm, 4 °C) and incubated for 1 h at 4 °C with glutathione-Sepharose (Thermo Scientific, IL, U.S.A). The sepharose was washed 3 times with IP lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1% Triton-X100, 1% NP40, 2mM EDTA, 0.1% Tween 20). HEK293T cells were transduced with different Flag-KPNA constructs. 48 hrs after transfection, cells were lysed with IP lysis buffer, clarified and pre-cleared with glutathione-Sepharose. Pre-cleared lysate was incubated with GST-NLS fragment bound to glutathione-Sepharose for 2 hrs at 4 °C. The sepharose was then washed 3 times with IP wash buffer (50mM Tris pH 7.4, 300mM NaCl, 1% Triton-X100, 1% NP40, 2mM EDTA, 0.1% Tween 20) and eluted by boiling in 1× SDS sample buffer for 5 mins at 95 °C.

Zhu F., Hwang B., Miyamoto S, & Rui L. (2016). Nuclear Import of JAK1 is Mediated by a Classical NLS and is Required for Survival of Diffuse Large B-cell Lymphoma. Molecular cancer research : MCR, 15(3), 348-357.

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GST-VRK2 Interaction Analysis

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IP lysis buffer containing protease and phosphatase inhibitors was used for lysis of pancreatic cancer cells. After centrifugation, the supernatant was collected, and 10 μg of the GST-VRK2 fusion protein was added for incubation overnight at 4°C. The next day, 40 μL of glutathione-Sepharose (Thermo, G2879) was added for another 4-hour incubation at 4°C. The beads were washed 3 times with wash buffer, and 1× loading buffer was then added for western blot analysis.

Chen J., Qiao K., Zhang C., Zhou X., Du Q., Deng Y, & Cao L. (2022). VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer. International Journal of Biological Sciences, 18(3), 1288-1302.

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Quantitative GST-Protein Binding Assay

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The concentration of purified recombinant proteins was measured using Bradford’s reagent (Bio-Rad, 5000202). About 0.25 to 2 μg of each protein was used in a binding reaction. Binding reactions were carried out in a binding buffer containing 25 mM Hepes pH 7.5, 12.5 mM MgCl2, 300 mM KCl, 0.1% NP-40, 1 mg/ml bovine serum albumin, 1 mM DTT. GST and His-tagged proteins were mixed in binding buffer to a total volume of 200 μl and incubated in a rotator for 1 h at 4 °C. Glutathione-sepharose (Thermo-Scientific, 25236) were washed thrice in binding buffer and then added to binding reactions at a packed bead volume of 10 μl per reaction. After another 1 h incubation at 4 °C with rotation, beads were washed thrice in washing buffer (25 mM Hepes pH 7.5, 12.5 mM MgCl2, 400 mM KCl, 0.5% NP-40) and eluted by boiling for 5 min in 2× SDS buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol). Samples were then analyzed by SDS-PAGE followed by Western blotting.

Ramakrishnan A.B., Burby P.E., Adiga K, & Cadigan K.M. (2022). SOX9 and TCF transcription factors associate to mediate Wnt/β-catenin target gene activation in colorectal cancer. The Journal of Biological Chemistry, 299(1), 102735.

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Influenza Virus Neuraminidase Activity Assay

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Rosetta 2 cells and SEC molecular weight standard were from Agilent. Imidazole, isopropyl β-D-1-thiogalactopyranoside (IPTG), 1 ml HisTrap Fast Flow crude columns, glutathione-sepharose, Tris, phosphate, citric acid, CaCl₂, protease inhibitor cocktail (PIN), Sf-900 III serum free medium (Sf-900), FreeStyle 293 (293F) cells, FreeStyle 293 expression media, simple blue stain, Novex WedgeWell 4 to 12% Tris-Glycine SDS-PAGE gels, Novex sharp unstained protein standard, Maxisorp 96-well plates, and BCA protein assay kit were obtained from Thermo Fisher Scientific. Centrifugal filters (10 and 30 kDa), polyethylenimine, and 4-methylumbelliferyl-α-D-N-acetylneuraminide (MUNANA) were acquired from Amicon, Polysciences, and Cayman Chemical, respectively. Bovine fetuin, 4-methylumbelliferone sodium salt (4-MU), o-phenylenediamine dihydrochloride, valproic acid, and HRP-linked peanut agglutinin were purchased from Sigma. Low protein binding 96-well black clear bottom fluorescent assay plates were from Corning. Specific-pathogen-free hen’s eggs were purchased from Charles River Labs. The reassortant Influenza A viruses WSN^H1N1/BR18 (N1 virus), which carries the HA and NA genes from the strain A/Brisbane/02/2018 (H1N1), and WSN^N2-KS17 (N2 virus), which carries the N2 gene from the strain A/Kansas/14/2017, were generated for prior studies (53 , 72 ).

Klenow L., Elfageih R., Gao J., Wan H., Withers S.G., de Gier J.W, & Daniels R. (2023). Influenza virus and pneumococcal neuraminidases enhance catalysis by similar yet distinct sialic acid–binding strategies. The Journal of Biological Chemistry, 299(2), 102891.

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Expression and Purification of Bromodomain Proteins

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Yeast Bdf1 bromodomain 1 (residues 132–263) or 2 (residues 317–430) and human Brd4 bromodomain 1 (residues 22–204) were cloned into pGEX-4T1 as GST-tagged proteins. Expression was induced in E. coli strain BL21 (DE3) grown in LB medium with kanamycin (50 μg/mL) at 37°C by adding 0.5 mM IPTG at OD₆₀₀ of 1 and then incubated for 16 h at 16°C. Cells were lysed by sonication in Tris-HCl 50 mM pH 7.5, NaCl 150 mM and protease inhibitors. Clarified lysate was incubated with glutathione sepharose (Fisher, ref. W7349W) and washed in Tris-HCl 50 mM pH 7.5, NaCl 500 mM, NP-40 1%. Bound proteins were eluted in glutathione 10 mM.

García-Oliver E., Ramus C., Perot J., Arlotto M., Champleboux M., Mietton F., Battail C., Boland A., Deleuze J.F., Ferro M., Couté Y, & Govin J. (2017). Bdf1 Bromodomains Are Essential for Meiosis and the Expression of Meiotic-Specific Genes. PLoS Genetics, 13(1), e1006541.

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GST-tagged SV2C-L4 Protein Binding

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GST-tagged SV2C–L4_(454–580) protein (100 μg) was incubated with 50 μl of glutathione Sepharose (Fisher Scientific, Ballycoolin, Ireland) for 1 h at 4 °C. After washing with 1 ml of binding buffer30 (link)32 (link), resin was incubated with different concentrations of rA, rAΔH_CN or rA(W₉₈₅L) DC in binding buffer. After washing 3 times with 1 ml of binding buffer, bound proteins were eluted by LDS sample buffer containing DTT with a final concentration of 50 mM. The reduced samples were analyzed by SDS-PAGE followed by Western blotting, using antibodies against LC/A or GST.

Wang J., Meng J., Nugent M., Tang M, & Dolly J.O. (2017). Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain. Scientific Reports, 7, 44474.

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