Glutathione agarose

Manufactured by Thermo Fisher Scientific

Sourced in United States

Glutathione agarose is a resin-based affinity chromatography medium used for the purification of glutathione-S-transferase (GST) fusion proteins. The agarose beads are covalently coupled with reduced glutathione, which serves as a ligand for the capture of GST-tagged recombinant proteins.

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Lab products found in correlation

Ni nta agarose, by Thermo Fisher Scientific (3 mentions) Gex4t 1 vector, by Merck Group (2 mentions) Amylose resin, by New England Biolabs (2 mentions) Ni sepharose 6 fast flow, by GE Healthcare (2 mentions) Coomassie brilliant blue staining, by Bio-Rad (2 mentions) Fla 3000 plus dage system, by Fujifilm (2 mentions) Ni nta his binding resin, by Thermo Fisher Scientific (2 mentions) Reduced glutathione, by Merck Group (2 mentions) Talon metal affinity resin, by Takara Bio (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Gst fusion protein, by Thermo Fisher Scientific (2 mentions) Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Anti flag m2 affinity gel, by Merck Group (2 mentions) Ni nta agarose, by Qiagen (2 mentions) Prescission protease, by GE Healthcare (2 mentions) Agarose, by Beyotime (1 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)

57 protocols using glutathione agarose

Immunoprecipitation and GST Affinity Isolation

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EECs were transfected for 48 h and incubated on ice with immunoprecipitation lysis buffer (Beyotime, P0013). For each sample, 500 μL of lysate was incubated with 2 μg of antibody and 800 μL of protein A/G plus agarose (Santa Cruz Biotechnology, sc-2003) overnight. The agarose beads were washed 4 times with 1 mL of lysis buffer containing 1% NP-40 (Beyotime, ST366). The precipitates were detected by SDS-PAGE and immunoblotting. For the GST affinity-isolation assays, GST or GST-HSPA1A protein was bound to glutathione agarose (Thermo Scientific, 21516) according to the manufacturer’s instructions, and the beads were washed four times. The beads were incubated with the target proteins harvested from the transfected EECs in affinity-isolation lysis buffer for 2 h at 4°C. The eluted proteins were detected by SDS-PAGE and immunoblotting.

Yang B., Xue Q., Guo J., Wang X., Zhang Y., Guo K., Li W., Chen S., Xue T., Qi X, & Wang J. (2019). Autophagy induction by the pathogen receptor NECTIN4 and sustained autophagy contribute to peste des petits ruminants virus infectivity. Autophagy, 16(5), 842-861.

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GST-Fhit Protein Pulldown Protocol

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In total, 150 μg of GST or GST-Fhit protein was bound to glutathione agarose (50% slurry; Thermo Scientific) following the manufacturer’s instructions. Equal amounts of protein lysate were added to the columns and rotated at 4 °C overnight. The resin was washed five times in a 1:1 mixture of TBS (50 mM Tris-Cl, 150 mM NaCl) and RIPA (EMD Millipore, Burlington, MA) buffers and eluted in 1× RSB buffer (2% SDS, 60 mM Tris-Cl, 5% glycerol, and 3% β-mercaptoethanol) for western blotting.

Druck T., Cheung D.G., Park D., Trapasso F., Pichiorri F., Gaspari M., Palumbo T., Aqeilan R.I., Gaudio E., Okumura H., Iuliano R., Raso C., Green K., Huebner K, & Croce C.M. (2019). Fhit–Fdxr interaction in the mitochondria: modulation of reactive oxygen species generation and apoptosis in cancer cells. Cell Death & Disease, 10(3), 147.

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GST-Fusion Protein Pulldown Assay

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Recombinant GST‐fusion proteins were expressed in E. coli BL21 (DE3) (Promega, L1195) using 0.5 mM IPTG at 25°C O/N. Bacteria were lysed in NET‐N buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P‐40) containing EDTA‐free protease inhibitor mixture (Sigma‐Aldrich, P8465) and subjected to sonication. GST proteins were purified and immobilized on glutathione agarose (Thermo Scientific, 16100). HA‐TFG^wtLIR or HA‐TFG^mutLIR3 or positive control (supplementary Fig 5E and F) vectors were transiently transfected in HeLa cells as indicated. Then, cells were lysed with NET‐N buffer. GST pulldown assays were performed by incubating immobilized GST‐fusion proteins with protein extract derived from HeLa cells for 2 h at 4°C with gentle agitation. Unbound proteins were removed by washing the resins five times with NET‐N buffer. GST proteins were eluted from the beads with 10 mM reduced glutathione in 50 mM Tris–HCl (pH 8.0), for 30 min with gentle agitation at room temperature. The eluted samples were boiled for 5 min in sample loading buffer and separated by SDS–PAGE.

Carinci M., Testa B., Bordi M., Milletti G., Bonora M., Antonucci L., Ferraina C., Carro M., Kumar M., Ceglie D., Eck F., Nardacci R., le Guerroué F., Petrini S., Soriano M.E., Caruana I., Doria V., Manifava M., Peron C., Lambrughi M., Tiranti V., Behrends C., Papaleo E., Pinton P., Giorgi C., Ktistakis N.T., Locatelli F., Nazio F, & Cecconi F. (2021). TFG binds LC3C to regulate ULK1 localization and autophagosome formation. The EMBO Journal, 40(10), e103563.

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Producing Polyclonal Antibodies for HyFat and HyDs

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Peptides corresponding to the C-terminal portions of HyFat (residues 4,191 to 4,393) and HyDs (residues 9,144 to 9,799) were expressed as GST fusions using the GEX4t-1 vector (Millipore) in Escherichia coli strain BL21 and purified on glutathione-agarose (Thermo Scientific). Purified proteins were used to immunize guinea pigs (Cocalico Biologicals) and rats (Covance). Polyclonal antibodies were purified by binding to antigen immobilized on glutathione-agarose beads and elution with 100 mM glycine (pH 2.5) into 1 M Tris, pH 8.0.

Brooun M., Klimovich A., Bashkurov M., Pearson B.J., Steele R.E, & McNeill H. (2020). Ancestral roles of atypical cadherins in planar cell polarity. Proceedings of the National Academy of Sciences of the United States of America, 117(32), 19310-19320.

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Purification and Interaction of ERK1/2 and PHD2

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His-tagged ERK1/ERK2 and GST-tagged PHD2 were expressed in E. coli BL21 treated with 0.1 mM IPTG at 16°C for 12 hr to induce protein expression. Bacteria were then harvested and resuspended in PBS containing 0.5% Triton X-100 and 1 mM PMSF, followed by ultrasonication for 20 min. The recombinant His-tagged and GST-tagged proteins were purified using Ni-NTA Agarose (25214, Thermofisher) and Glutathione Agarose (16100, Thermofisher) respectively. For the ERK1/2 and PHD2 GST pull-down assay, GST-tagged PHD2 was incubated with purified ERK1 or ERK2 at 4°C for 2 hr. The elution was analyzed by western blot with indicated antibodies (24 (link)).

Li Z., Zhou W., Zhang Y., Sun W., Yung M.M., Sun J., Li J., Chen C.W., Li Z., Meng Y., Chai J., Zhou Y., Liu S.S., Cheung A.N., Ngan H.Y., Chan D.W., Zheng W, & Zhu W. (2019). ERK regulates HIF-1α-mediated platinum resistance by directly targeting PHD2 in ovarian cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(19), 5947-5960.

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Purification of Recombinant Kinesin Proteins

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Rosetta™ host bacteria derived from BL21 carrying pGEX-4T-1-KIF5B-C wild-type or mutants were cultured with 100μg/ml ampicillin at 37°C till the optical density (OD) at wavelength of 600 reached 0.6, and then induced for protein expression with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C overnight. After centrifuged at 5,000 rpm and re-suspended in buffer A (50 mM Tris, 150 mM NaCl, pH8.0, 0.01% Triton X-100, 4 mM DTT, plus protease inhibitor cocktail), the bacteria were lysed by French pressure cell press. The supernatants from centrifugation at 18,000 rpm for 30 minutes were incubated with the glutathione agarose (Thermo Fisher) for 1 hr at 4°C. The protein-agarose mixture was loaded onto a plastic chromatography column and washed with buffer A for 3 times. The protein was eluted by the elution buffer (50 mM Tris, 150 mM NaCl, pH8.0, 10 mM reduced glutathione, 0.01% Triton X-100) and dialyzed witthe binding buffer (300 mM NaCl, 20 mM Tris, pH7.4). The purity of the purified proteins was examined by SDS-PAGE and Coomassie blue staining.

Wang Z., Zhang F., He J., Wu P., Tay L.W., Cai M., Nian W., Weng Y., Qin L., Chang J.T., McIntire L.B., Di Paolo G., Xu J., Peng J, & Du G. (2017). Binding of PLD2-generated phosphatidic acid to KIF5B promotes MT1-MMP surface trafficking and lung metastasis of mouse breast cancer cells. Developmental cell, 43(2), 186-197.e7.

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Purification of Recombinant GST-GmSPL9d, MBP-GmWUSa, and MBP-GmWUSb

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To purify recombinant GST‐GmSPL9d, MBP‐GmWUSa and MBP‐GmWUSb, the open‐reading frames of GmSPL9d, MBP‐GmWUSa and MBP‐GmWUSb were cloned into pGEX‐4T‐1 and pMAL‐c2x. The GST‐GmSPL9d and MBP‐GmWUSa expression plasmids were then transformed into Escherichia coli strain BL21. Protein purification was performed using glutathione agarose (Thermo Fisher Scientific, Waltham, MA) and amylose resin (NEB, Ipswich, MA) according to the manufacturers' instructions. The primers used to construct the plasmids are listed in Table S2.

Sun Z., Su C., Yun J., Jiang Q., Wang L., Wang Y., Cao D., Zhao F., Zhao Q., Zhang M., Zhou B., Zhang L., Kong F., Liu B., Tong Y, & Li X. (2018). Genetic improvement of the shoot architecture and yield in soya bean plants via the manipulation of GmmiR156b. Plant Biotechnology Journal, 17(1), 50-62.

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Protein-Protein Interaction Analysis

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The recombinant plasmids were transformed into BL21(DE3)pLysS chemically competent cells. SlIAA9-GST was purified with Glutathione Agarose (Thermo Fisher Scientific, United States) according to the company manual instruction. MBP, MBP-SlARF6A, MBP-SlARF8A, MBP-SlARF8B, and MBP-SlARF24 were purified as fusion proteins immobilized with amylose resin (New England Biolabs, United States) following standard protocols. Five micrograms of GST-SlIAA9 protein were pre-incubated with 10 μL pre-washed amylose resin in 150 μL incubation buffer (1 mM NaCl, 20 mM MgCl₂, 0.2% Triton X-100, and 0.1 M HEPES at pH7.2) for 1 h at 4°C. The resin was collected by centrifugation and washed five times with washing buffer (20 mM Tris-HCl at pH 7.5, 300 mM NaCl, 0.1 mM EDTA, 0.5% Triton-X100). The pull-down proteins were detected by western blot with an α-GST antibody (Thermo Fisher Scientific, United States). All primer sequences used in this analysis are listed in Supplementary Table S2.

Wu L., Tian Z, & Zhang J. (2018). Functional Dissection of Auxin Response Factors in Regulating Tomato Leaf Shape Development. Frontiers in Plant Science, 9, 957.

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GST Pulldown Assay for Protein-Protein Interactions

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Recombinant proteins were expressed in E. coli BL21 (DE3) cells with 0.5 mM isoprophyl-β-D-thiogalactoside (IPTG) at 16 °C overnight. Trx-His and GST-tagged proteins were purified with Ni–nitrilotriacetic acid (Ni-NTA) agarose (Thermo Fisher Scientific) and glutathione agarose (Thermo Fisher Scientific), respectively. For the GST pulldown assay, equal amounts of recombinant GST and GST-NIb were incubated with Trx-His or Trx-His-NPR1 and GST beads at 4 °C for 2 h. After washing with wash buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.5], 0.05% NP-40) six times, the GST beads were boiled for 5 min in 2× SDS‒PAGE loading buffer. Equal amounts of supernatants were loaded onto a 10% acrylamide gel, separated by electrophoresis, and detected with antibodies against GST or 6×Histidine.

Liu J., Wu X., Fang Y., Liu Y., Bello E.O., Li Y., Xiong R., Li Y., Fu Z.Q., Wang A, & Cheng X. (2023). A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity. Nature Communications, 14, 3580.

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Purification of GST-tagged Proteins

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GST-tagged proteins used for in vitro binding assays were expressed in E. coli Rosetta (DE3)pLysS cells. After 3 h induction with 0.5 mM IPTG at 37°C, cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM DTT, pH 8.0 + 1× Roche cOmplete protease inhibitor cocktail) and lysed by sonication. After centrifugation to pellet cell debris, the GST-tagged fusion proteins were purified from the lysate using glutathione agarose (Thermo Scientific) according to the manufacturer's instructions. Purified protein was concentrated and buffer-exchanged to 20 mM HEPES–KOH, 100 mM KCl, 1.5 mM MgCl₂, 5% glycerol, pH 8.0.

Norppa A.J., Kauppala T.M., Heikkinen H.A., Verma B., Iwaï H, & Frilander M.J. (2018). Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA. RNA, 24(3), 396-409.

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