Affinity Sepharose Purification of MRTF-A

Preparation of affinity Sepharose covalently coupled with each of the stereoisomers of CCG-1423 was performed based on previously reported methods [13 (link)]. MRTF-A protein was synthesized in vitro using the TNT SP6 High-Yield Expression System based on an optimized wheat germ extract (Promega) and was purified using anti-Flag M2 affinity gel. Mixtures of MRTF-A protein (300 ng), 0.005% bovine serum albumin, and the indicated CCG-1423 Sepharose or control Sepharose (bed volume 25 μl) in the pull-down (PD) buffer [13 (link)] (total 400 μl)] were incubated at 4°C for 2 h with rotation. After washing the respective Sepharose with the PD buffer and phosphate-buffered saline, the pull-downed MRTF-A protein was detected by IB.