HEK293T cells were transfected with Flag-tagged or GST-Tagged genes of interest. Cells were harvested at 48 h post transfection, and lysed with Triton X-100 buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 20 mM β-glycerophosphate, 10% glycerol) supplemented with a protease inhibitor cocktail (Roche). Whole cell lysates (WCLs) were sonicated, incubated at 4°C for 30 min on a rotator, and centrifuged at 12,000 rpm for 30 min. Supernatant was filtered, precleared with sepharose 4B agarose beads (Thermo) at 4°C for 1 h. The pre-cleared WCLs were incubated with anti-FLAG M2 agarose beads or glutathione-conjugated agarose beads at 4°C for 4 h. Anti-FLAG M2 magnetic beads were washed extensively with lysis buffer and eluted with 0.2 mg/ml 3xFlag peptide. GST beads were extensively washed and used immediately for in vitro on-column deamidation assay. Concentration of purified proteins was analyzed by SDS-PAGE and Coomassie staining, with BSA as a standard.
Sepharose 4b agarose beads
Manufactured by Thermo Fisher Scientific
Sepharose 4B agarose beads are a type of chromatography resin used for the separation and purification of biomolecules. They are composed of agarose, a polysaccharide derived from seaweed, and have a bead-like structure. Sepharose 4B beads are designed to provide a high-resolution and efficient matrix for the separation of various biological molecules, such as proteins, enzymes, and nucleic acids.
Automatically generated - may contain errors
2 protocols using sepharose 4b agarose beads
1
Affinity Purification of Tagged Proteins
2
Affinity Purification of RIG-I and Nsp5 Proteins
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HEK293T cells were transfected with plasmids containing Flag-tagged RIG-I and RIG-I-(11–925) for 36 h. WCLs were prepared with Triton X-100 buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 20 mM β-glycerophosphate, 10% glycerol) supplemented with a protease inhibitor cocktail (Roche). WCLs were sonicated, rotated at 4°C for 20 min, and centrifuged at 12,000 rpm and 4°C for 15 min. The supernatant was filtered and precleared with Sepharose 4B Agarose beads (Thermo) at 4°C for 1 h. The precleared samples were incubated with anti-Flag M2 Agarose beads at 4°C for 4 h. Agarose beads were washed extensively with lysis buffer. Flag-tagged proteins were eluted with 0.2 mg/ml 3×Flag peptide.
Escherichia coli BL21(DE3) was transformed with pGEX-4T-1 or the pET28 plasmid containing RIG-I-2CARD, Nsp5, or Nsp5-C145A. The expression of recombinant GST-tagged protein was induced by 0.1 mM IPTG (isopropyl-β-d -thiogalactopyranoside) at 20°C. Bacteria were harvested, lysed, and incubated with glutathione-conjugated agarose (GE) for 4 h at 4°C. Agarose beads were washed extensively, and GST-tagged proteins were eluted with 10 mM reduced glutathione.
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