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Pull-Down Assay

MS Min Sui
MX Meimei Xiong
YL Yuling Li
QZ Qiao Zhou
XS Xiaofei Shen
DJ Da Jia
MG Maling Gou
QS Qingxiang Sun
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All proteins used were purified by S200 prior to pull-down. To assess different interactions, we immobilized GST-tagged proteins (0.5 n mol) on GSH beads. Soluble proteins at indicated concentrations were incubated with the immobilized proteins in a total volume of 1 mL for 2 h at 4 °C. After two washing steps, bound proteins were separated by SDS/PAGE and visualized by Coomassie Blue staining. Each experiment was repeated at least twice and checked for consistency. Pull-down buffer contains 20 mM Tris pH 7.5, 200 mM NaCl, 10% glycerol, 2 mM MgCl2, 0.005% Triton-X100 and 2 mM DTT.

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