Gst sepharose beads

Manufactured by GE Healthcare

Sourced in United States

GST Sepharose beads are an affinity chromatography resin used for the purification of glutathione S-transferase (GST) fusion proteins. The beads consist of cross-linked agarose with covalently attached glutathione, which binds to the GST tag on the target protein, allowing for selective capture and purification.

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Lab products found in correlation

Ni sepharose beads, by GE Healthcare (3 mentions) Hispur ni nta spin column, by Thermo Fisher Scientific (1 mentions) Protein g agarose, by Thermo Fisher Scientific (1 mentions) Egfp c1 vector, by Takara Bio (1 mentions) E coli bl21 de3, by New England Biolabs (1 mentions) Instantblue protein stain, by Abcam (1 mentions) Hiload 16 600 superdex 200 pg column, by GE Healthcare (1 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Tnt system, by Promega (1 mentions)

Close spelling variants of this product

GST-Sepharose 4B beads GST-Sepharose GST beads Sepharose beads

13 protocols using gst sepharose beads

Purification of GST-WDFY2 and His-MBP-FYVE Domains

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GST-tagged WDFY2 was expressed in E. coli Rosetta2(DE3) cells (Novagen, Madison, WI). Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 10 μM ZnCl2, 2.5 mM 2-mercaptoethanol) and purified using GST sepharose beads (GE Healthcare). Purified protein was eluted in elution buffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol, 10 μM ZnCl₂, 2.5 mM 2-mercaptoethanol, and 10 mM reduced glutathione)^{51 (link)} and directly used in protein–lipid overlay assays.
His-MBP-tagged 2xFYVE domains derived from HRS and WDFY2 were purified by Ni-NTA affinity chromatography. Recombinant protein was expressed in Rosetta2(DE3), purified using HisPur Ni-NTA spin columns (Thermo Fisher) and dialyzed against liposome buffer (50 m Hepes, 150 mM KCL, 100 µM ZnCl2, 1 mM TCEP). Purified protein was flash-frozen in small aliquots and stored at −80 °C.

Sneeggen M., Pedersen N.M., Campsteijn C., Haugsten E.M., Stenmark H, & Schink K.O. (2019). WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling. Nature Communications, 10, 2850.

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Kinase assay for p62 and ULK1

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Glutathione S-transferase (GST)-tagged recombinant p62 and GST-ULK1 proteins were purified from bacteria using GST Sepharose beads according to the manufacturer's instruction (GE Healthcare). GST-p62 cDNAs were kindly provided by Dr. T. Johansen (University of Tromsø, Norway). GST-ULK1 cDNAs were kindly provided by Dr. S-W Yu (Daegu Gyeongbuk Institute of Science and Technology [DGIST], Republic of Korea). HEK293 cells transfected with MYC-PERK WT, MYC-PERK kinase dead (K618A) were subjected to immunoprecipitation using a mouse anti-MYC antibody for 4 h at 4 °C, followed by overnight incubation at 4 °C with G-Sepharose beads. The next day, the kinase reaction was initiated by treating with 200 μM ATP and recombinant GST-p62 or GST-ULK1 proteins for 40 min at 37 °C. The kinase reaction buffer was composed of 20 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT, and 0.4 mM NaF at pH 7.2–7.5. The reaction was terminated by adding sodium dodecyl sulfate sample buffer and boiling for 10 min at 100 °C. The samples were subjected to immunoblot analysis using anti-phospho-p62 (S351) and anti-phospho-ULK1 (S317).

Lee D.H., Park J.S., Lee Y.S, & Bae S.H. (2022). PERK prevents hepatic lipotoxicity by activating the p62-ULK1 axis-mediated noncanonical KEAP1-Nrf2 pathway. Redox Biology, 50, 102235.

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Purification and Immunoprecipitation of MKRN1 and AMPK

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Glutathione S-transferase (GST)-tagged recombinant MKRN1 and the AMPKα1, α2, β1 and γ1 proteins were purified from bacteria using GST Sepharose beads according to the manufacturer’s protocol (GE Healthcare).
Immunoprecipitation assay: The cells were lysed in lysis buffer (50 mM of Tris-HCl (pH 7.5), 150 mM of NaCl, 0.5% Triton X-100 and 1 mM of EDTA) containing a protease inhibitor cocktail. The cell lysates were then incubated with 1 µg of antibody with rotation, followed by incubation with 25 µl of protein G agarose (Invitrogen), and the precipitated proteins were eluted in SDS sample buffer under boiling conditions^{53 (link)}.

Lee M.S., Han H.J., Han S.Y., Kim I.Y., Chae S., Lee C.S., Kim S.E., Yoon S.G., Park J.W., Kim J.H., Shin S., Jeong M., Ko A., Lee H.Y., Oh K.J., Lee Y.H., Bae K.H., Koo S.H., Kim J.W., Seong J.K., Hwang D, & Song J. (2018). Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation. Nature Communications, 9, 3404.

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Purification and Analysis of Seckel Syndrome Protein

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The region deleted in the Seckel syndrome patient (residues 1,073–1,159 of CPAP) was cloned into the pGEX vector (Amersham Biosciences) containing an N‐terminal GST tag and the eGFP‐C1 vector (Clontech). The protein was expressed in E. coli BL21(DE3) (New England Biolabs) and induced by 0.5 mM IPTG overnight at 18°C in LB medium. The recombinant protein was affinity‐purified from supernatant using GST‐sepharose beads (GE Healthcare). The conjugated protein was then treated with HeLa cell extracts and further analyzed by Western blots.

Gabriel E., Wason A., Ramani A., Gooi L.M., Keller P., Pozniakovsky A., Poser I., Noack F., Telugu N.S., Calegari F., Šarić T., Hescheler J., Hyman A.A., Gottardo M., Callaini G., Alkuraya F.S, & Gopalakrishnan J. (2016). CPAP promotes timely cilium disassembly to maintain neural progenitor pool. The EMBO Journal, 35(8), 803-819.

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GST-Sepharose Affinity Purification

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GST-Sepharose beads (20 μl, GE Healthcare) was incubated with bacterial lysates with GST or GST-Raf fusion proteins in a final volume of 300 μl of buffer A (1 mM DTT, 20% glycerol, 50 mM Na₂H₂PO₄ pH 8.0, 300 mM NaCl and 1 mM PMSF) at 4 °C for an hour with rotation, washed 5 times with 0.8 ml of buffer A with 1% Triton X-100 and once with 0.8 ml of buffer B (10 mM Tris pH 7.5, 1 mM EDTA, 210 mM NaCl, 15% glycerol, 0.25% NP-40, 1 mM DTT, 1 mM Na₃VO₄, 1 mM PMSF and 1X protease inhibitor cocktail. Beads were then reacted with a 300 μl mixture containing cell lysates, 1X protease inhibitor cocktail, 1 mM DTT and 1 mM PMSF at 4 °C for hour with rotation, washed 3 times with buffer B, boiled for 7 min in 1X SDS sample buffer, and then subjected to immunoblotting analysis.

Yeh Y.H., Hsiao H.F., Yeh Y.C., Chen T.W, & Li T.K. (2018). Inflammatory interferon activates HIF-1α-mediated epithelial-to-mesenchymal transition via PI3K/AKT/mTOR pathway. Journal of Experimental & Clinical Cancer Research : CR, 37, 70.

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PERK-Mediated Phosphorylation of eIF2α

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eIF2α was phosphorylated on residue Ser51 using purified PERK kinase, as described above. 1 mg of purified PERK, in a final volume of 1 ml, was incubated with 50 μl of GST Sepharose beads (GE Healthcare), pre-equilibrated with kinase buffer for 30 min at RT. Excess PERK was removed by washing the beads 3 times with 1 ml kinase buffer, by spinning the samples gently at 2,000 r.p.m. for 5 min at 4 °C. 500 μg of purified eIF2α, in a final volume of 1 ml, predialyzed in kinase buffer, was added to PERK-containing GST beads. 5 mM ATP (pH 7.4) was added to the reaction and phosphorylation was allowed to proceed for 1 h at 37 °C with shaking at 350 r.p.m. The supernatant, containing phosphorylated eIF2α, was collected. Phosphorylated eIF2α was further purified by size exclusion on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using dephosphorylation buffer (50 mM Tris (pH 7.4), 1.5 mM EGTA (pH 8.0), 2 mM MnCl₂). Elution fractions were run on 4–12% NuPAGE Bis-Tris gels (Life Technologies) and visualized by staining with InstantBlue Protein Stain (Expedeon). Samples containing pure phosphorylated eIF2α (P-eIF2α) were pooled and concentrated to 5 mg/ml, and stored at −80 °C in small aliquots.

Carrara M., Sigurdardottir A, & Bertolotti A. (2017). Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors. Nature structural & molecular biology, 24(9), 708-716.

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Recombinant Expression and Purification of AnxA2 Fragments

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AnxA2 protein was amplified using a cDNA clone (Origene RC205081) and cloned into the pGEX4T3 vector with a N-terminal GST tag. A series of 12 Anxa2 fragments (detailed in Fig. S4) were expressed in a similar manner. Protein expression was induced using 1 mM IPTG at 22 °C for 16 h. Expressed proteins were extracted and sonicated in lysis buffer containing 1 M Tris (pH 8.0), 300 mM NaCl, 0.1% TritonX-100, 2 mM PMSF and protease inhibitor cocktail (Roche). Lysate was clarified by centrifugation at 13,200 rpm at 4 °C for 30 min and the supernatant purified with GST-Sepharose beads (GE biosciences). Sequences of primers used for generation of these mutants are listed in Supplementary Data 2.

Kalra R.S., Soman G.S., Parab P.B., Mali A.M., Varankar S.S., Naik R.R., Kamble S.C., Dhanjal J.K, & Bapat S.A. (2021). A monoclonal antibody against annexin A2 targets stem and progenitor cell fractions in tumors. Translational Oncology, 15(1), 101257.

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GST Fusion Protein Binding Assay

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The GST alone and GST fusion proteins, including GST- ERα 29-180aa and GST- ERα 282-595aa 37 (link), were expressed in BL21 bacteria and bound to GST-Sepharose beads according to the manufacturer's instructions (GE Healthcare). The expression plasmid for Flag-MDC1 was used for synthesis Flag-MDC1 protein in Vitro with transcription and translation in the TNT system (Promega). Equal amounts of GST alone, or GST-fusion proteins coupled with GST-Sepharose beads were incubated with in vitro-translated Flag-MDC1 protein at 4°C overnight. The precipitated proteins were washed thoroughly 4 times with binding buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol, 1% Nonidet P-40). The bound proteins were detected by western blot and stained by Coomassie Brilliant Blue.

Zou R., Zhong X., Wang C., Sun H., Wang S., Lin L., Sun S., Tong C., Luo H., Gao P., Li Y., Zhou T., Li D., Cao L, & Zhao Y. (2015). MDC1 Enhances Estrogen Receptor-mediated Transactivation and Contributes to Breast Cancer Suppression. International Journal of Biological Sciences, 11(9), 992-1005.

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Protein Expression and Purification

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The pET28a and pGEX-4T-1 vectors were used as backbones to construct plasmids for His- and GST-tagged protein expression, respectively. Primers used are listed in Supplementary Table 2. His- and GST-tagged recombinant proteins were expressed in Escherichia coli strain BL21 (DE3), and purified by using Ni-Sepharose beads (GE Healthcare) and GST Sepharose beads (GE Healthcare), respectively. Purified GST or GST fusion protein was immobilized on glutathione Sepharose beads in binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 10% glycerol, protease inhibitors), and then incubated with His fusion protein for 4 h at 4 °C. After incubation, the beads were washed with binding buffer five times, loading buffer was added, and the beads were boiled for 10 min. The samples were then subjected to SDS-PAGE and analyzed by western blotting with monoclonal anti-His antibody.

Yan H., Chen C., Chen H., Hong H., Huang Y., Ling K., Hu J, & Wei Q. (2020). TALPID3 and ANKRD26 selectively orchestrate FBF1 localization and cilia gating. Nature Communications, 11, 2196.

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LASP1 Protein Interactions by Immunoprecipitation and GST-pulldown

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2×10⁶ MDA-MB-231-shLASP1 control cells were lysed in RIPA+-buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 % sodium-deoxycholate, 1 % Triton-X-100, 0.1 % SDS, 1 mM sodium orthovanadate, 10 mM sodium-pyrophosphate, 10 mM EDTA and protease inhibitor mixture). Immunoprecipitation was performed by rotation of 500 μl cell lysate with 2 μg His₆-LASP1 for 2 h at 4°C followed by incubation with magnetic His-beads for 15 min at 4°C. For GST-pulldown, 500 μl cell lysate was incubated with 2 μg GST-tagged LASP1 for 1 h at 4°C followed by incubation with 50 μl GST-sepharose beads (GE Healthcare) for 60 min. The immune complexes were washed two times with ice-cold PBS buffer and prepared for SDS-PAGE.

Endres M., Kneitz S., Orth M.F., Perera R.K., Zernecke A, & Butt E. (2016). Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1). Oncotarget, 7(39), 64244-64259.

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