GST and GST fusion proteins were expressed in Escherichia coli BL-21 cells by induction with a final concentration of 0.8 mM isopropyl-b-D-thiogalactopyranoside. Cells were lysed by sonication in 10 ml of 1X PBS (NaCl/Pi) supplemented with complete protease inhibitor tablets (Roche Applied Science, Basel, Switzerland). GST fusion proteins with ER-α36 and ER-α66 were purified using glutathione-agarose beads (Sigma-Aldrich; Merck KGaA). The recombinant human His-tag-PRMT2 protein (obtained from Yingrun Biotechnologies Inc.) were mixed with 10 mg of GST derivatives bound to glutathione-agarose beads in 0.5 ml of binding buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3 mM dithiothreitol, 0.1% NP-40 and protease inhibitor tablets from Roche Applied Science). The binding reaction was performed at 4°C for 3 h and the beads were subsequently washed 4 times with the washing buffer (the same as the binding buffer), 30 min each time. The beads were eluted by boiling in SDS sample buffer and analyzed by SDS/PAGE, and PRMT2, ER-α36 and ER-α66 were analyzed by immunoblotting.
Glutathione agarose beads
Manufactured by Roche
Glutathione-agarose beads are a type of affinity chromatography resin used for the purification of proteins that contain a glutathione S-transferase (GST) tag. The beads consist of agarose particles to which glutathione, a tripeptide, is covalently attached. Proteins with a GST tag bind to the glutathione on the beads, allowing for their separation from other proteins in a sample.
Automatically generated - may contain errors
2 protocols using glutathione agarose beads
1
Purification of GST-fusion Proteins
2
Phosphorylation of CATs by PC1
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To obtain phosphorylated CATs, WT (Kitaake) seedlings at the 3-leaf stage were treated with 140 mM NaCl for 30 min, and the phosphorylated CATs proteins were immunoprecipitated with an anti-CAT antibody. Then, recombinant GST-PC1 or GST purified from E. coli with glutathione agarose beads (Roche, CH) was added to an in vitro phosphate reaction buffer [50 mM HEPES, pH 7.4, 50 mM NaCl, 0.1% (v/v) Triton X-100, and 1 mM DTT] with a different metal ion (5 mM EDTA, 10 mM MgCl 2 , or 10 mM MnCl 2 ) for 90 min at 30 °C to verify the ion requirements for PC1. The phosphorylation state of CATs was determined by immunoblotting using antiphospho-serine (pSer, Abcam, ab9332, dilution 1:1,000), anti-phospho-threonine (pThr, Abcam, ab9337, dilution 1:1,000), or anti-phospho-tyrosine (pTyr, Abcam, ab17302, dilution 1:1,000) antibodies.
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