Co-Immunoprecipitation (Co-IP)

ZL Zhaozheng Li
YZ Yao Zhao
ZP Zhenwei Pan
BC Benzhi Cai
CZ Chengwei Zhang
JJ Jundong Jiao
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VSMC were washed three times with PBS and fully lysed on ice with cell lysis buffer containing protease inhibitors for 30 min. The lysates were centrifuged at 11,000 g for 15 min at 4 °C, and the supernatants were collected. SRPK1 antibody (5ug) or control mouse immunoglobulin G (IgG) (5ug) was added to the protein samples and incubated at 4 °C overnight. The prepared Protein A/G-MagBeads suspension was added to the protein samples and shaken in a vertical mixer at 4 °C for 2 h. The protein samples were denatured and eluted in an eluent in a boiling water bath for 10 min. Magnetic beads were adsorbed using a magnetic holder, and the sample supernatant was collected for the next step of the experiment.

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