Glutathione sepharose bead

Manufactured by GE Healthcare

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Glutathione-Sepharose beads are a chromatography resin designed for the purification of glutathione-S-transferase (GST) fusion proteins. The beads consist of cross-linked agarose beads with covalently coupled glutathione, which can selectively bind to GST-tagged proteins. This allows for the isolation and enrichment of GST-fusion proteins from complex mixtures.

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Lab products found in correlation

Prescission protease, by GE Healthcare (17 mentions) Protease inhibitor cocktail, by Roche (9 mentions) Reduced glutathione, by Merck Group (8 mentions) Ni nta agarose, by Qiagen (7 mentions) Amylose resin, by New England Biolabs (6 mentions) Ni nta agarose bead, by Qiagen (5 mentions) Thrombin, by GE Healthcare (5 mentions) Tnt t7 coupled reticulocyte lysate system, by Promega (4 mentions) Lysozyme, by Merck Group (4 mentions) Protease inhibitor, by Merck Group (4 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions) Anti flag m2 affinity gel, by Merck Group (3 mentions) Tnt rabbit reticulocyte system, by Promega (3 mentions) Ni sepharose beads, by GE Healthcare (3 mentions) Tnt t7 quick coupled transcription translation system, by Promega (3 mentions) Ni beads, by GE Healthcare (3 mentions) Pgex 2t vector, by GE Healthcare (3 mentions) Pgex 4t 1, by GE Healthcare (3 mentions) Glutathione sepharose 4b beads, by GE Healthcare (3 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Promega (2 mentions)

Close spelling variants of this product

Glutathione sepharose (246 citations) Glutathione beads (75 citations) Sepharose beads (43 citations) Glutathione Sepharose 4B bead slurry (4 citations) Glutathione–Sepharose 4B bead column (2 citations)

494 protocols using glutathione sepharose bead

Purification and GST pull-down of recombinant proteins

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Both the ceWRC and hWRC were recombinantly expressed and purified essentially as previously described (Chen et al., 2014b (link)). GST-tagged HPO-30 ICD and TIAM-1 PDZ were expressed in BL21 (DE3) T1^R cells at 18 °C overnight and purified using glutathione sepharose beads (GE Healthcare). MBP-tagged HPO-30 ICD and DMA-1 ICD were similarly expressed and purified using amylose beads (New England Biolabs). The tagged ICDs and PDZ domain were further purified using Source 15Q and/or Source 15S ion exchange columns (GE Healthcare) before use.
GST pull-down was performed as previously described (Chen et al., 2014a (link)). Briefly, 130 to 300 pmol of GST-tagged bait and 130 to 300 pmol of prey proteins were mixed with 20 µL of glutathione sepharose beads (GE Healthcare) in 1 mL of pulldown buffer (20 mM HEPES pH 7, 50 mM NaCl, 5% (w/v) glycerol and 5 mM β-mercaptoethanol) at 4 °C for 30 min. After three washes using 1 mL of the pulldown buffer, bound proteins were eluted with GST elution buffer (100 mM Tris-HCl pH 8.5, 50 mM NaCl, 5% (w/v) glycerol, 5 mM β-mercaptoethanol and 30 mM reduced glutathione) and examined by SDS-PAGE.

Zou W., Dong X., Broederdorf T.R., Shen A., Kramer D.A., Shi R., Liang X., Miller DM I.I.I., Xiang Y.K., Yasuda R., Chen B, & Shen K. (2018). A dendritic guidance receptor complex brings together distinct actin regulators to drive efficient F-actin assembly and branching. Developmental cell, 45(3), 362-375.e3.

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Protein-protein Interaction Assays

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For in vivo binding assays, cells were collected and lysed as above in lysis buffer lacking SDS and NP40. Yeast lysates were incubated with glutathione-Sepharose beads (GE Healthcare) for two hours. Beads were washed extensively with the same buffer, resuspended in SDS loading buffer, and proteins were analysed by SDS-PAGE and immunoblotting as described [12] (link). In vitro binding assays were performed by mixing E. coli extracts containing GST or GST-fused proteins with E. coli extracts bearing the corresponding His-tagged proteins, and then processed as above.
Purification of Pmk1-HA6H and Pmp1-GST fusions was performed with Ni²⁺-NTA-agarose beads (Qiagen) and glutathione-Sepharose beads (GE Healthcare), respectively, as described previously [20] (link).

Sacristán-Reviriego A., Madrid M., Cansado J., Martín H, & Molina M. (2014). A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts. PLoS ONE, 9(1), e85390.

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Recombinant H3-H4-ASF1a Complex Binding

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The recombination methods of the H3-H4 tetramer and the H3-H4-ASF1a complex in vitro were shown in Supplementary Methods^{12 (link),53 (link)}. About 50 μg protein, GST, GST-TRIM66-WT_965-1160, or its mutant (D986A, N1123A, and D986A/N1123A) were bound to glutathione-sepharose beads (GE Healthcare) in a buffer (20 mM Tris, 200 mM NaCl, and 1 mM DTT) at pH 7.5 and incubated at 4 °C for 4 h. The glutathione-sepharose beads were pelleted and washed thrice with washing buffer. Subsequently, the beads were incubated with 100–150 μg recombinant H3K56ac-H4-ASF1a complex in a buffer containing 20 mM Tris, 150 mM NaCl, and 1 mM DTT at pH 7.5 for 6 h at 4 °C. The beads were pelleted and washed thrice using the same buffer. The captured proteins were eluted and analyzed using SDS-polyacrylamide gel electrophoresis (PAGE).
For the GST-pulldown assays of the TRIM66-WT_965-1160 against the calf thymus histones, the beads with about 50 μg GST-TRIM66-WT_965-1160 were incubated with 50 μg calf thymus histones in a buffer containing 20 mM Tris, 500 mM NaCl, and 1 mM DTT at pH 7.5 for 1 h at 4 °C. The beads were pelleted and washed five times using the same buffer. The captured proteins were eluted and analyzed using SDS-PAGE.

Chen J., Wang Z., Guo X., Li F., Wei Q., Chen X., Gong D., Xu Y., Chen W., Liu Y., Kang J, & Shi Y. (2019). TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells. Nature Communications, 10, 4273.

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GST Pull-Down Assay for Protein-Protein Interactions

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GST pull down assay was referred to the previous report^{26 (link)}. Specifically, the 5 μg purified Flag-Nsun2 (mouse) protein was pre-cleared with glutathione Sepharose beads (Cat. no. 17075601; GE Healthcare) in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 7.4), 0.5% NP-40) at 4 °C for 1 h. Then pre-cleared Flag-Nsun2 was mixed with GST or GST-RoRγt protein and incubated at 4 °C for 4 h with equal amount of pre-cleared glutathione Sepharose beads. The complex of IPed proteins and beads was washed five times with NETN buffer, then heated with 4×NuPAGE LDS Buffer (Cat. no. NP0007; Invitrogen) at 95 °C for 10 min, and analyzed by western blot. The plasmids information was listed in Supplementary Data 6 and antibodies were listed in Supplementary Data 5.

Yang W.L., Qiu W., Zhang T., Xu K., Gu Z.J., Zhou Y., Xu H.J., Yang Z.Z., Shen B., Zhao Y.L., Zhou Q., Yang Y., Li W., Yang P.Y, & Yang Y.G. (2023). Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis. Nature Communications, 14, 863.

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Recombinant Protein Interaction Assay

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GST, GST-MoCka1, GST-MoCkb1, GST-MoCkb2 and His-MoRgs1 were expressed in Escherichia coli BL21-CodonPlus (DE3) cells (Sigma, CMC0014). Cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF [Beyotime Biotechnology, ST506-2]) with a sonicator (Branson). Samples were centrifuged (13,000 g, 10 min) and the supernatants were transferred to a new 1.5 ml tube and stored at 70°C. The GST, GST-MoCka1, GST-MoCkb1 and GST-MoCkb2 supernatants were then mixed with 30 ml glutathione sepharose beads (GE Healthcare, 10265165) and incubated at 4°C for 2 h. The recombinant GST, GST-MoCka1, GST-MoCkb1, and GST-MoCkb2-bound to glutathione sepharose beads were incubated with E. coli cell lysate containing His-MoRgs1 at 4°C for another 4 h. Finally, the beads were washed with buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF, 1% Triton X-100) five times and eluted from the beads. Eluted proteins were then analyzed by immunoblot (IB) with monoclonal anti-His and monoclonal anti-GST antibodies, respectively.

Yu R., Shen X., Liu M., Liu X., Yin Z., Li X., Feng W., Hu J., Zhang H., Zheng X., Wang P, & Zhang Z. (2021). The rice blast fungus MoRgs1 functioning in cAMP signaling and pathogenicity is regulated by casein kinase MoCk2 phosphorylation and modulated by membrane protein MoEmc2. PLoS Pathogens, 17(6), e1009657.

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Purification of OutB Periplasmic Domain

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DNA sequence encoding the conserved periplasmic domain of D. dadantii OutB was cloned into the pGEX-6P-3 vector (GE Healthcare) encoding a cleavable N-terminal Glutathione S-transferase (GST) affinity tag (Table S4). Three GST-OutB constructs encoding OutB residues 112-220, 112-202 and 112-192 were expressed in E. coli BL21 (DE3) cells (Stratagene). The bacteria were grown in Luria broth supplemented with 100 µg/ml ampicillin to an OD600 of 0.6 next, induced with 1 mM isopropyl-β-Dthiogalactopyranoside (IPTG) and grown for an additional 16 h at 20°C. The cells were suspended in phosphate buffered saline (PBS) containing 0.01 M phosphate buffer, 0.0027 M KCl and 0.137 M NaCl, pH 7.4 and lysed by sonication. Cell debris was removed by centrifugation at 35,000g for 20 min and the supernatant from 1 l of cell culture was incubated with 2.5 ml of Glutathione Sepharose beads (GE Healthcare) at 4°C for 6 h The Glutathione Sepharose beads were then collected in a PD10 column and washed with 50 ml of PBS. GST tag was on column cleaved by addition of 20 µl of (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted August 6, 2021. ; https://doi.org/10.1101/2021.08.06.455404 doi: bioRxiv preprint

Zhang S., Gu S., Rycroft P., Ruaudel F., Delolme F., Robert X., Ballut L., Pickersgill R.W., & Shevchik V.E. (2021). Scaffolding protein GspB/OutB facilitates assembly of the Dickeya dadantii type 2 secretion system by anchoring the outer membrane secretin pore to the inner membrane and to the peptidoglycan cell wall.

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Protein Induction and Purification

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For protein induction, Escherichia coli BL21 cells transformed with pGEX-4T-1 or pGEX-4T-1-PKM2 were incubated at 20 °C overnight. LB broth (5 mL) was then added in a 15 mL conical tube. Transfected cells were induced using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Promega, Madison, WI, USA) and adjusted to an A₆₀₀ of 0.4–0.5. Cells were lysed by sonication and incubated with glutathione-sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Purified proteins were rotated with HEK293T cell lysates overexpressing Myc-HAUSP at 4 °C overnight, and bound proteins were analyzed by western blotting.

Choi H.S., Pei C.Z., Park J.H., Kim S.Y., Song S.Y., Shin G.J, & Baek K.H. (2020). Protein Stability of Pyruvate Kinase Isozyme M2 Is Mediated by HAUSP. Cancers, 12(6), 1548.

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Purification and Interaction of GST-USP7 with Flag-Raf-1

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For protein induction, Escherichia coli BL21 cells transformed with pGEX-4T-1 vector or pGEX-4T-1-USP7 were incubated at 20 °C overnight. LB broth (5 mL) was then added in a 15 mL conical tube. Transfected cells were induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Cat #V3955, Promega, Madison, WI, USA) and adjusted to an A600 of 0.4–0.5. Cells were lysed by sonication and incubated with glutathione-sepharose beads (Cat #27-4574-01, Pharmacia Biotech, Uppsala, Sweden). Purified proteins were rotated with HEK293T cell lysates overexpressing Flag-Raf-1 at 4 °C overnight, and bound proteins were analyzed by Western blotting.

Park H.B., Hwang S, & Baek K.H. (2022). USP7 regulates the ERK1/2 signaling pathway through deubiquitinating Raf-1 in lung adenocarcinoma. Cell Death & Disease, 13(8), 698.

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Investigating FOXM1-PMLIV Protein Interactions

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GST‐FOXM1 fusion constructs were expressed in BL21‐Star™ (DE3) pLysS cells (Thermo Fisher Scientific), and crude bacterial lysates were prepared by sonication in cold lysis buffer (50 mm Tris/HCl pH 8.0, 100 mm NaCl, 1 mm EDTA, pH 8.0, 2% NP‐40, 1 mm DTT, 1 mm PMSF). To test the interaction between FOXM1 and PMLIV, GST‐fusion proteins were freshly purified by glutathione‐Sepharose beads (GE Healthcare, Chicago, IL, USA), washed two times with lysis buffer and one time with GST‐Wash buffer (300 mm KCl, 20 mm HEPES pH 7.9, 0.1% NP40, 5 mm MgCl₂), and resuspended in 200 μL GST‐interaction buffer (150 mm KCl, 20 mm HEPES pH 7.9, 0.1% NP40, 5 mm MgCl₂) and mixed with 200 μg of HEK‐293T cell lysate overexpressing (OE) mRED‐PMLIV. The binding reaction was incubated for 3 h at 4 °C. Beads were washed three times with GST‐Wash buffer (600 mm KCl, 20 mm HEPES pH 7.9, 0.1% NP40, 5 mm MgCl₂) and resuspended in SDS sample buffer. Samples were subjected to SDS/PAGE and analyzed by immunoblotting.

Sachini N., Arampatzi P., Klonizakis A., Nikolaou C., Makatounakis T., Lam E.W., Kretsovali A, & Papamatheakis J. (2019). Promyelocytic leukemia protein (PML) controls breast cancer cell proliferation by modulating Forkhead transcription factors. Molecular Oncology, 13(6), 1369-1387.

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Affinity Pulldown of FITC-Labeled Peptides

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Pull-down of 10 µM of FITC-labeled peptides (Table S2) with 5 µM of purified GST-SH3 domains (Table S1) was performed using 10 µL of glutathione Sepharose beads (GE Healthcare, Chalfont Saint Giles, UK) in a buffer containing 30 mM Tris-HCl at pH 7.5, 3 mM dithiothreitol, and 5 mM MgCl2 for 1 h at 4 °C. Purified GST was used as a negative control. After three washes, bound proteins were eluted by incubation in the same buffer containing 20 mM reduced glutathione for 15 min at 4 °C, and the beads were separated by centrifugation. Bound FITC-labeled peptides were detected by dot blot analysis using 1 µL of eluent at an emission wavelength of 600 nm and an Odyssey Fc imaging system (LI-COR Biosciences, Lincoln, NE, USA). Detected signals were quantified densitometrically using LI-COR Image Studio version 5.2 imaging software.

Kazemein Jasemi N.S., Mehrabipour M., Magdalena Estirado E., Brunsveld L., Dvorsky R, & Ahmadian M.R. (2024). Functional Classification and Interaction Selectivity Landscape of the Human SH3 Domain Superfamily. Cells, 13(2), 195.

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