Ip matrix

Four micrograms normal rabbit IgG (Beyotime, China, A7016) or IP antibody, 40 μl suspended IP Matrix (Santa Cruz, USA, sc45039) and 500 μl PBS were incubated at 4 °C on a rotator for at least an hour. The mixture was then centrifuged and washed three times with PBS in the presence of a protease inhibitor mixture (protease inhibitor, phosphatase inhibitor, PMSF; KangChen, Shanghai, China) and then discard supernatant carefully. Cells that transfected for 48 h were lysed and transferred to the matrix, incubating at 4 °C on a rotator overnight. Next, the matrix was centrifuged and washed for five times. SDS-PAGE sample loading buffer (Beyotime, China, P0015) was added to the immunoprecipitates followed by boiling for 10 min at 100 °C. The IP and input proteins were detected by western blot.

Co-IP assays were performed as we previously described [28 (link)]. In brief, cell lysates were prepared using NP-40 buffer (50mM Tris-HCl pH 7.5, 1% NP-40, 150mM NaCl, 5 mM EDTA) and incubated with IP matrix (Santa Cruz) conjugated to IP antibody (anti-HA, Santa Cruz; or anti-Flag M2, Sigma) for overnight. The IP matrix-antibody complex was then washed with NP-40 buffer, and protein complexes were eluted and subjected to Western-blot assays.

Rat posterior pituitary lysates were precleared with protein A/G Sepharose (GE Healthcare) or pre-clearing matrix (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 4°C. Pre-cleared lysates were incubated with 10 μg of control IgG or IP antibodies at 4°C followed by additional incubation with protein A/G Sepharose for 3 h at 4°C. Protein A Sepharose was used for mouse IP antibodies and protein G Sepharose was use for rabbit IP antibodies. The IP matrix (Santa Cruz Biotechnology) was incubated with 5 μg of control IgG (1 μg) or IP antibodies overnight at 4°C followed by additional incubation with pre-cleared lysates overnight at 4°C. Thereafter, the sediments were washed four times with the wash buffer used in the in vitro binding assays, and bound proteins were eluted in SDS sample buffer.