Recombinant GST fusion proteins were expressed in E. coli, and purified on glutathione-agarose 4B (Peptron) according to the manufacturer's instructions. His-tagged proteins were also produced in E. coli and purified on Ni-NTA beads (Invitrogen) according to the manufacturer's guidelines. Typical SUMOylation reactions were conducted in a 30 µl volume containing 70 nM GST-SAE2/SAE1, 1 µM His-Ubc9, and 9 µM His-SUMO-1GG or GST-SUMO-1GG in buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 1 mM DTT, and 5 mM ATP). To prepare flag-PIAS1 protein, 293T cells in a 150-mm dish were transfected with 30 µg of flag-PIAS1-expressing plasmid, followed by immunoprecipitation of total cell lysates with 50 µl of anti-flag M2 antibody. SUMOylation reaction mixes were incubated for 1 h at 37°C. After terminating the reaction with SDS sample buffer containing β-mercaptoethanol, the reaction products were fractionated by SDS-PAGE.
Glutathione agarose 4b
Manufactured by Peptron
Glutathione-agarose 4B is an affinity chromatography resin designed for the purification of glutathione-S-transferase (GST)-tagged recombinant proteins. The resin consists of glutathione immobilized on a 4% cross-linked agarose bead support, which allows for the specific capture and separation of GST-fusion proteins from complex samples.
Automatically generated - may contain errors
4 protocols using glutathione agarose 4b
1
Purification and SUMOylation Assay Protocol
2
Protein-Protein Interaction Assay Protocol
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MBP-CDF2, MBP-DCL1-RBD and MBP were expressed in E. coli BL21 (DE3) and purified according to the manufacturer’s protocol (New England Biolabs). Glutathione–agarose 4B (Peptron) beads were used to purify GST-CDF2, GST-DCL1-RBD and GST. The partially purified recombinant GST and MBP fusion proteins were bounded to glutathione–agarose beads and amylose resin respectively, MBP-DCL1-RBD was incubated with the GST-CDF2 captured by glutathione sepharose beads, whereas GST-DCL1-RBD was incubated with the MBP-CDF2 bound to amylose resin. The GST and MBP proteins were performed in parallel assays as negative controls, mixed with total protein extracts in 1mL pull-down buffer (40 mM HEPES-KOH, pH 7.5, 10 mM KCl, 3 mM MgCl2, 0.4 M sucrose, 1 mM EDTA, 1 mM DTT, and 0.2% Triton X-100), and then incubated at 4°C for 1 h with agitation [49 (link)]. The beads were washed four times with the binding buffer. Proteins were eluted and further analyzed by immunoblotting using appropriate antibodies.
3
In vitro Methylation and Binding Assays
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In vitro methylation assays were performed as described previously (Bedford et al., 2000 (link)). In brief, GST-PRMT1 (mouse), PRMT3 (mouse), PRMT4 (mouse), PRMT6 (Rat), and various GST-Smurf2 constructs were purified using Glutathione-Agarose 4B (Peptron). Various GST-Smurf2 constructs (0.5 μg) were incubated with indicated GST-PRMTs (0.2–1.0 μg) in the presence of 2 μl of S-adenosyl-L-[methyl-3H] methionine ([3H]AdoMet; 83.3 ci/mmol, Perkin Elmer) for the indicated time at 30°C in 20 μl of methylation reaction buffer (15 mM Tris-HCl (pH.7.5), 25 mM NaCl 10 mM EDTA). Methylation reactions were stopped by the addition of 4X SDS sample buffer, followed by heating 100°C for 10 min. Samples were separated on SDS-PAGE and stained with 0.05% Coomassie Brilliant Blue. After destaining, gels were soaked in EN3HANCE (PerkinElmer) according to the manufacturer’s instructions and visualized by fluorography after exposure for 1 day to 1 week at −80°C.
For in vitro binding assay, bacterially expressed GST-Smurf2 were purified using glutathione-agarose beads (GE Healthcare) and incubated with [S35]-labelled Myc-PRMT1, which was generated by an in vitro translation system (Invitrogen). After immunoprecipitation, the samples were analyzed by SDS-PAGE, followed by immunoblotting.
For in vitro binding assay, bacterially expressed GST-Smurf2 were purified using glutathione-agarose beads (GE Healthcare) and incubated with [S35]-labelled Myc-PRMT1, which was generated by an in vitro translation system (Invitrogen). After immunoprecipitation, the samples were analyzed by SDS-PAGE, followed by immunoblotting.
4
Protein Interaction Assay Protocol
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The cDNAs of PIF4 and DCL1 were amplified and subcloned to the pMAL-c2x and pGEX4T-1 plasmids, and confirmed by sequencing. Primers used to amplify the genes are listed in S3 Table . MBP-DCL1-RBD and MBP were expressed in E. coli BL21 (DE3) and purified according to the manufacturer’s protocol (New England Biolabs). GST-PIF4 and GST were purified using glutathione–agarose 4B (Peptron) beads. For protein pull-down assays, GST-PIF4 was incubated with the MBP-DCL1-RBD bound to amylose resin, mixed with total protein extracts in 1ml protein pull-down buffer (40 mM HEPES-KOH, pH 7.5, 10 mM KCl, 3 mM MgCl2, 0.4 M sucrose, 1 mM EDTA, 1 mM DTT, and 0.2% Triton X-100), and then incubated at 4°C for 1 h with agitation. GST and MBP proteins were used as negative controls in parallel assays. The beads were washed four times with the binding buffer. Proteins were eluted and further analyzed by immunoblotting using appropriate antibodies.
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