Fig 1: Tandem affinity purification of chromatin-remodeling complex protein Baf57c using SGTAP.(A) Schematic of LR recombination reaction used to create pEpic CMV:Baf57c-SGTAP. (B) Schematic of steps for TAP of Baf57-SGTAP. Step 1: Baf57c with a C-terminally conjugated SBP, TEV protease cleavage site, and tandem copies of protein G is first isolated by affinity purification using IgG-sepharose beads; Step 2: Baf57c-SBP is cleaved from protein G bound to IgG-sepharose beads by the addition of TEV protease; Step 3: Baf57c-SBP is further isolated by affinity purification using streptavidin-sepharose beads; Step 4: Baf57c-SBP is finally eluted from streptavidin by the addition of biotin. (C) Western blot for SBP at various stages of Baf57c purification from nuclear extracts of HEK293T cells expressing pEpic CMV-Baf57c-SGTAP. 10% of each indicated fraction was used for immunoblotting. Lane 1: the crude nuclear extract; Lane 2: nuclear extract after incubation with IgG beads; Lane 3: post-TEV protease cleavage of proteins bound to IgG beads; Lane 4: SDS elution of proteins from beads following Streptavidin purification. The asterisk indicates Baf57c-SGTAP fusion proteins; the arrow indicates the cleaved Baf57c-SBP fusion; molecular weights in kilodaltons are shown at the right.