E. coli cells (BL21DE3) expressing GST-tagged anti-GFP nanobody (Addgene plasmid #61838) [28 (link)] were suspended in 1X PBS (137 mM NaCl, 2.7 mM KCl, 100 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4) containing 1 mM PMSF, 1 mM EDTA, 1 mM DTT, 10 mg/ml lysozyme and 1% Triton X-100 and kept on ice for 20 min. Cells were then sonicated, and cell lysates containing GST-tagged anti-GFP nanobodies were incubated with glutathione resin (G-Biosciences, U.S.A.) at 4 °C for 1 h. Nanobody-bound glutathione resins were harvested by brief centrifugation followed by washes with 1X PBS containing 1 mM PMSF. GST-tagged anti-GFP nanobody-bound glutathione resins were incubated with yeast (GS115) whole-cell protein lysate containing GFP expressing PAOX1, PGDH2 or PPEPCK at 4 °C for 2–3 h. Post-interaction, resins were centrifuged briefly and washed at least thrice with cold 1X PBS. Bound proteins were resolved by SDS‒PAGE and visualized by Coomassie Brilliant Blue R-250 staining.
Glutathione resin
Manufactured by G Biosciences
Sourced in United States
Glutathione resin is a chromatography media used for the purification of proteins that have an affinity for glutathione. It consists of glutathione, a tripeptide composed of glutamic acid, cysteine, and glycine, that is coupled to a solid support matrix. The resin can be used to selectively capture and purify proteins or enzymes that possess a glutathione S-transferase (GST) tag, which binds to the immobilized glutathione.
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Lab products found in correlation
4 protocols using glutathione resin
1
Affinity Purification of GFP-Binding Nanobodies
2
Nanobody-Based Protein Interaction Assay
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E. coli cells (BL21DE3) expressing GST tagged anti-GFP nanobody (Addgene plasmid #61838) (Katoh et al., 2015) (link) were suspended in 1X PBS (137 mM NaCl, 2.7 mM KCl, 100 mM Na2HPO4 and 2 mM KH2PO4, pH-7.4) containing 1 mM PMSF, 1 mM EDTA, 1 mM DTT, 10g/ml lysozyme and 1% Triton X-100 and kept on ice for 20 min. Cells were then sonicated and cell lysate containing GST tagged anti-GFP nanobody was incubated with glutathione resin (G-Biosciences, U.S.A.) at 4ºC for 1 h. Nanobody bound glutathione resins were harvested by brief centrifugation followed by washes with 1X PBS containing 1 mM PMSF. GST tagged anti-GFP nanobody bound glutathione resins were incubated with yeast (GS115) whole cell protein lysate containing GFP expressing from either PAOX1, PGDH2 or PPEPCK at 4ºC for 2-3 h. Post interaction resins were centrifuged briefly and washed atleast thrice with cold 1X PBS. Bound proteins were resolved by SDS-PAGE and visualized by Coomassie Brilliant Blue R staining.
3
Purification and Binding of GST-Chs3 Exomer Proteins
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GST-Chs3 fragment constructs were constructed in the pGEX-2T vector (GE Healthcare) and transformed into Rosetta2 (DE3) E. coli cells (Novagen) for expression. 1 L culture was grown to ~3 OD600 in TB media at 37°C, temperature lowered to 18°C, then expression induced with 240 uM IPTG. After overnight expression, cells were harvested by centrifugation, resuspended in 50 ml PBS buffer with 1 mM DTT, and lysed by sonication. GST fusion proteins were isolated by adding 100 μl equilibrated glutathione resin (G-Biosciences) to 5 ml cleared lysate and incubating with rotation at least 2 h at 4°C. Resin was washed 3 times with 1 ml PBS+DTT and resuspended in 500 μl PBS+DTT. 10 μl of 5 mg/ml exomer protein was added and mixture was incubated ~1 h at 4°C. Resin was washed 3 times with 1 ml PBS+DTT and analyzed by SDS-PAGE and Western blot with anti-6xHis antibody (Covance).
4
Purification of GST-tagged Ypt1 Protein
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GST‐Ypt1‐7xHis (gift from T. Bretscher laboratory) or GST‐Ypt1 (pLT50, (Thomas & Fromme, 2016 )), with a cleavable N‐terminal GST tag in the pGEX‐6P vector backbone, was transformed into the Rosetta2 strain of E. coli (Novagen). 1–4 l of culture were grown in terrific broth for 8–12 h at 37°C (until the OD600 ~2–3). The temperature was then reduced to 16°C and 1 h later IPTG was added to a final concentration of 300 µM to induce protein expression. Following overnight expression (14–18 h), cells were collected via centrifugation and resuspended in lysis buffer (1× PBS, 5 mM BME, 2 mM MgCl2). Cells were lysed by sonication, and the clarified lysate was incubated with Glutathione resin (G‐Biosciences) for 2–3 h at 4°C to isolate GST‐tagged proteins. The resin was washed in lysis buffer, resuspended in PreScission cleavage buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM MgCl2, and 1 mM DTT), and treated overnight with PreScission (3C) protease (~40 µl at 1.3 mg/ml) at 4°C to remove the GST tag. Cleavage of the GST tag eluted the proteins from the resin. After treatment, the supernatant was collected and analyzed by SDS–PAGE.
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