Glutathione sepharose beads

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Glutathione sepharose beads are a solid-phase affinity chromatography medium used for the purification of glutathione S-transferase (GST) fusion proteins. These beads are composed of agarose beads conjugated with reduced glutathione, which enables the binding and capture of GST-tagged proteins.

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Pd 10, by GE Healthcare (2 mentions) Cam sepharose 4b, by GE Healthcare (1 mentions)

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Glutathione beads (9 citations) Glutathione sepharose (3 citations)

8 protocols using glutathione sepharose beads

EGFR Protein Expression and Purification

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A construct encoding EGFR residues 696–1022 with a GST tag was cloned into baculovirus expression vector pAcG2T. The protein was expressed by infecting SF9 cells with high titer viral stocks for 48 h. Cells were harvested and lysed in 25 mM Tris (pH 7.9), 150 mM NaCl, and 1 mM DTT. The supernatant was incubated with glutathione Sepharose beads (Genscript). After washing with wash buffer (40 mM Tris pH 7.9, 500 mM NaCl, 1% Glycerol, 1 mM DTT), the beads were incubated overnight with 5ml wash buffer containing 5ul of 5 mg/ml alpha-thrombin to remove GST tag. The eluted EGFR protein was loaded on desalt column PD-10 (GE) to change the buffer to 25 mM Tris pH7.5, 50 mM NaCl, 20 mM MgCl2, and 1 mM DTT. The protein was concentrated to 1 mg/ml and aliquots were frozen and stored at −80C.

Wu H., Wang A., Zhang W., Wang B., Chen C., Wang W., Hu C., Ye Z., Zhao Z., Wang L., Li X., Yu K., Liu J., Wu J., Yan X.E., Zhao P., Wang J., Wang C., Weisberg E.L., Gray N.S., Yun C.H., Liu J., Chen L, & Liu Q. (2015). Ibrutinib selectively and irreversibly targets EGFR (L858R, Del19) mutant but is moderately resistant to EGFR (T790M) mutant NSCLC Cells. Oncotarget, 6(31), 31313-31322.

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Glutathione Affinity Purification of In Vitro Proteins

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GST fusion proteins were bound to glutathione sepharose beads (Genscript, Nanjing, China) and incubated with in vitro translated protein at 4°C for 2 h with gentle agitation in binding buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.2% NP-40). After extensive washing with binding buffer, bound proteins were analyzed by Western blotting.

Yu J., Hu S., Ma K., Sun L., Hu H., Zou F., Guo Q., Lei Z., Zhou D., Sun Y., Zhang D., Ma L., Shen B, & Zhu C. (2014). Ribosomal Protein S29 Regulates Metabolic Insecticide Resistance through Binding and Degradation of CYP6N3. PLoS ONE, 9(4), e94611.

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GST-Pol ι Pulldown Assay

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To perform GST pulldown assays, GST and GST fusion Pol ι proteins were synthesized in E. coli BL21(DE3). GST-tagged Pol ι from BL21 was immobilized on glutathione sepharose beads (GenScript, L00206). GST or GST fusion proteins were mixed with MYC-USP7 and HA-HIF-1α expressed HEK293T cell lysates. The eluted proteins were analyzed by western blot.

Gao A., Zhang M., Zhu S.Q., Zou S., Chen H., Li X., He C., Zhou L., Mei Y., Ding W., Zhou J., Zhou Y, & Cao Y. (2024). DNA polymerase iota promotes EMT and metastasis of esophageal squamous cell carcinoma by interacting with USP7 to stabilize HIF-1α. Cell Death & Disease, 15(2), 171.

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Coprecipitation of GFP-Cobl-like Proteins

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Coprecipitation experiments with extracts from HEK293 cells expressing different GFP-Cobl-like proteins were essentially done as described before (Qualmann et al., 1999 (link); Schwintzer et al., 2011 (link)). In brief, HEK293 cell lysates were incubated for 3 h at 4°C with purified, recombinant GST-fusion proteins immobilized on glutathione sepharose beads (GenScript). The reactions were washed several times with lysis buffer containing 150 mM NaCl and EDTA-free protease inhibitor Complete. Bound protein complexes were eluted either with 20 mM reduced glutathione, 120 mM NaCl, and 50 mM Tris-HCl, pH 8.0 (30 min RT), or by boiling the beads in 4× SDS sample buffer.
For coprecipitations with CaM, HEK293 cell lysates were prepared in an EGTA-free lysis buffer containing 150 mM NaCl, EDTA-free protease inhibitor cocktail, and 200 µM calpain I inhibitor. Cell lysates were supplemented with 1 mM EGTA or 500 µM CaCl₂. After incubation with 25 µl CaM-sepharose 4B (GE Healthcare) for 3 h at 4°C and washing, bound proteins were isolated by boiling in SDS sample buffer.
Lysates, supernatants, and eluates were analyzed by immunoblotting using anti-GST and anti-GFP antibodies, respectively.

Izadi M., Schlobinski D., Lahr M., Schwintzer L., Qualmann B, & Kessels M.M. (2018). Cobl-like promotes actin filament formation and dendritic branching using only a single WH2 domain. The Journal of Cell Biology, 217(1), 211-230.

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Coprecipitation of GFP Fusion Proteins

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For coprecipitation experiments, extracts from an HEK293 cell expressing different GFP fusion proteins were incubated for 3 h at 4°C with purified GST fusion protein immobilized on glutathione Sepharose beads (GenScript). GST fusion protein binding, lysate incubation, and washing were performed in lysis buffer with 150 mM NaCl. Bound protein complexes were eluted with 20 mM reduced glutathione, 120 mM NaCl, and 50 mM Tris/HCl, pH 8.0, and analyzed by anti-GFP and anti-GST immunoblotting. Every experiment included samples with only GST instead of GST fusion proteins attached to the matrix to control for specificity and was conducted several times to reproduce data.

Schneider K., Seemann E., Liebmann L., Ahuja R., Koch D., Westermann M., Hübner C.A., Kessels M.M, & Qualmann B. (2014). ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function. The Journal of Cell Biology, 205(2), 197-215.

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Coprecipitation of GFP-Fusion Proteins

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For coprecipitation experiments, extracts from HEK293 cells expressing different GFP fusion proteins were incubated for 3 h at 4°C with purified GST-fusion proteins immobilized on glutathione sepharose beads (GenScript) as described [33 (link)]. Bound protein complexes were eluted with 20 mM-reduced glutathione, 120 mM NaCl, 50 mM Tris/HCl pH 8.0.
For coprecipitations with CaM, HEK293 cell lysates were prepared in EGTA-free lysis buffer containing 150 mM NaCl, and EDTA-free protease inhibitor cocktail and 200 μM calpain inhibitor I. Cell lysates were supplemented with either 1 mM EGTA or to be tested Ca²⁺ concentrations.
For binding curves, Ca²⁺ concentrations ranging from 0 to 500 μM were set according to [41 (link)]. After incubation with 25 μl CaM-sepharose 4B for 3 h at 4°C and washing, bound proteins were isolated by boiling in SDS sample buffer.
Lysates, supernatants, and eluates were analyzed by immunoblotting using anti-GST and anti-GFP antibodies, respectively.
For binding curves, quantitative immunoblotting experiments (n ≥ 3) were conducted and data expressed as percent binding, %(Elution/∑Elution)/(Elution/∑Elution+Supernatant/∑Supernatant) with pEGFP control values subtracted. Sigmoidal dose-response curves were fit from Graphpad Prism and modified using Adobe Illustrator.

Hou W., Izadi M., Nemitz S., Haag N., Kessels M.M, & Qualmann B. (2015). The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin. PLoS Biology, 13(9), e1002233.

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Purification of EGFR Protein from Baculovirus

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A construct encoding EGFR residues 696–1022 with a GST tag was cloned into baculovirus expression vector pAcG2T. The protein was expressed by infecting SF9 cells with high titer viral stocks for 48 h. Cells were harvested and lysed in 25 mM Tris (pH 7.9), 150 mM NaCl, and 1 mM DTT. The supernatant was incubated with glutathione Sepharose beads (Genscript). After washing with wash buffer (40 mM Tris pH 7.9, 500 mM NaCl, 1% Glycerol, 1 mM DTT), the beads were incubated overnight with 5 ml wash buffer containing 5 ul of 5 mg/ ml alpha-thrombin to remove GST tag. The eluted EGFR protein was loaded on desalt column PD-10(GE) to change the buffer to 25 mM Tris pH7.5, 50 mM NaCl, 20 mM MgCl2, and 1 mM DTT. The protein was concentrated to 1 mg/ml and aliquots were frozen and stored at −80°C.

Wang A., Yan X.E., Wu H., Wang W., Hu C., Chen C., Zhao Z., Zhao P., Li X., Wang L., Wang B., Ye Z., Wang J., Wang C., Zhang W., Gray N.S., Weisberg E.L., Chen L., Liu J., Yun C.H, & Liu Q. (2016). Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation. Oncotarget, 7(43), 69760-69769.

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Purification of GST-PIAS1 Fusion Protein

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pGEX4T1-PIAS1 was transformed into E. coli Rosetta. A single colony was inoculated in LB medium with 100 μg/ml ampicillin and cultured at 37 °C until the culture reached exponential phase. Expression of GST fusion proteins was induced by 100 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) for 10 h at 16 °C. The cells were pelleted and lysed by sonication in Buffer A (1 × PBS buffer containing 10% glycerol, 1 × protease inhibitor cocktail, and 1 mM DTT). After centrifugation at 10,000 rpm for 15 min, the supernatants were incubated with Glutathione Sepharose beads (GenScript) for 4 h at 4 °C. The GST fusion protein-bound beads were washed three times with cold Washing Buffer (1 × PBS buffer containing 0.05% Triton X-100) and were ready for in vitro pulldown experiments.

Chen Z., Zhang Y., Guan Q., Zhang H., Luo J., Li J., Wei W., Xu X., Liao L., Wong J, & Li J. (2021). Linking nuclear matrix–localized PIAS1 to chromatin SUMOylation via direct binding of histones H3 and H2A.Z. The Journal of Biological Chemistry, 297(4), 101200.

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