Protein a sepharose cl 4b column

Immunoprecipitation and immunoblotting analyses were performed as previously described (30 (link)). Briefly, tissues or cells were homogenized in a buffer containing 50 mM tris-HCl (pH 7.5), 0.5% Triton X-100, 0.15 M NaCl, 4 mM EDTA, 4 mM EGTA, 1 mM Na₃VO₄, 50 mM NaF, 1 mM dithiothreitol, and protease inhibitors (trypsin inhibitor, pepstatin A, and leupeptin). For immunoprecipitation, lysates were incubated for 2 hours at 4°C with the indicated antibodies on a Protein A Sepharose CL-4B column (GE Healthcare Life Sciences) in homogenization buffer. The immunoprecipitates were washed thrice with homogenization buffer; equivalent amounts of protein were separated with SDS-PAGE, and proteins were then transferred to an Immobilon polyvinylidene difluoride membrane. Membranes were blocked with Tris Buffered Saline with Tween®20 (TBST) solution [50 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] containing 5% fat-free milk powder for 1 hour at room temperature and then incubated overnight at 4°C with the indicated primary antibodies. The membranes were then washed and incubated with the appropriate HRP-conjugated secondary antibody diluted in TBST. Blots were developed using an ECL immunoblotting detection system (Amersham Biosciences). Immunoreactive bands were visualized using the luminescent image analyzer LAS-4000 (Fujifilm).