Pre equilibrated glutathione sepharose 4b

Manufactured by GE Healthcare

Pre-equilibrated Glutathione Sepharose 4B is a pre-prepared affinity chromatography medium designed for the purification of glutathione S-transferase (GST) fusion proteins. The matrix consists of Sepharose 4B beads with covalently coupled glutathione, a ligand that binds to the GST tag. This ready-to-use format simplifies the purification process by eliminating the need for prior buffer equilibration.

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Lab products found in correlation

Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions) Reduced glutathione, by Merck Group (1 mentions) Zeba column, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Glutathione Sepharose 4B (822 citations) Glutathione sepharose (246 citations) Sepharose 4B (96 citations) Pre-equilibrated glutathione sepharose 4B resin (3 citations)

3 protocols using pre equilibrated glutathione sepharose 4b

GST Protein Interaction Assay

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GST or GST-AvrBsT were expressed in E. coli BL21-CodonPlus(DE3) cells (Stratagene). Cells were lysed in buffer (1X PBS, pH 8, 1% Triton X-100, 0.1% 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride (Sigma Aldrich)) with a sonicator (Branson). GST and GST-AvrBsT supernatants were incubated with 30 µL of pre-equilibrated Glutathione Sepharose 4B (GE Healthcare) for 1 h at 4°C with rotation. Sepharose beads were recovered by centrifugation and then washed with buffer for 5 min at 4°C with rotation. GST or GST-AvrBsT (WT, C222A, or K282R) bound beads were incubated with soluble E. coli lysates containing His6-ACIP1 for 2 h at 4°C with rotation. The beads were washed with buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 0.1% Triton X-100, and 0.1% 2-mercaptoethanol) three times. Protein bound to the beads was separated by SDS-PAGE and analyzed by immunoblot analysis. Anti-GST and anti-His sera were used to detect GST-AvrBsT and His6-ACIP1.

Cheong M.S., Kirik A., Kim J.G., Frame K., Kirik V, & Mudgett M.B. (2014). AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity. PLoS Pathogens, 10(2), e1003952.

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PTEN Protein Expression and Purification

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PTEN protein was expressed and purified as described (Redfern et al., 2008 (link)). Human PTEN with a C-terminal His-tag was expressed in E. coli BL21 (DE3). For the SAXS experiments, the His-tag was cleaved off using enterokinase. The GST pull-down assay was carried out using a batch method (results, see Supplementary Figure 1). Purified GST-PTEN and PTEN-His₆ were mixed in an equimolar ratio and allowed to incubate a bed of pre-equilibrated glutathione Sepharose 4B (GE Healthcare Life Sciences) for an hour at 4 °C on a rocker. As a negative control, GST protein and PTEN-His₆ were mixed using the same protocol. The resin was washed with buffers containing 0.5% triton X-100, 0.1% triton X-100, and finally detergent-free wash buffer. The remaining protein was eluted using 10 mmol/L reduced glutathione in Tris at pH 8.0. The eluted fractions were analyzed by SDS-PAGE. To confirm the presence of PTEN-His₆, a Western blot was carried out using a His-tag specific antibody.

Heinrich F., Chakravarthy S., Nanda H., Papa A., Pandolfi P.P., Ross A.H., Harishchandra R.K., Gericke A, & Lösche M. (2015). The PTEN tumor suppressor forms homodimers in solution. Structure (London, England : 1993), 23(10), 1952-1957.

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Recombinant Lpg2411 Purification Protocol

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Lpg2411 was produced as a GST fusion protein in E. coli BL21 (DE3) at 25°C overnight after induction with 0.5 mM isopropyl-β-dithiogalactopyranoside (IPTG). E. coli cells producing GST-Lpg2411 were harvested and resuspended in PBS supplemented with 1 mM MgCl 2 and 1 mM β-mercaptoethanol (PBS-MM) followed by lysis using the LV10 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (which this version posted July 9, 2021. ; https://doi.org/10.1101/2021.07.08.451723 doi: bioRxiv preprint 21 microfluidizer (Microfluidics). Cell lysate was centrifuged at 24,000 × g for 35 min, and the supernatant was incubated with pre-equilibrated glutathione sepharose 4B (GE Healthcare) for 2 hours at 4°C. The resin was washed three times with PBS-MM, and proteins were eluted in 50 mM Tris-HCl (pH 8) containing 10 mM reduced glutathione (Sigma). Glutathione was removed using a desalting Zeba column following manufacturer instructions (ThermoFisher Scientific).

Noll R.R., Pike C.M., Lehman S.S., Williamson C.D., & Neunuebel M.R. (2021). Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection.

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