Flag tag protein ip assay kit with magnetic beads

Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700, Millipore, USA) was used for RIP experiments in accordance with previously mentioned manufacturer’s instructions [39 (link)]. Cell lysates were incubated with 5 µg of CyPA primary antibodies (sc-134,310, Santa Cruz Biotechnology, USA). Mouse immunoglobulin (IgG) was used as a control. Western blot and qRT-PCR were used to detect the final product.
IP experiments were used with the IP Kit with Protein A + G Magnetic Beads (P2179S, Beyotime Biotechnology, China) and the Flag-Tag Protein IP Assay Kit with Magnetic Beads (P2181S, Beyotime Biotechnology, China). In Brief, the pretreated magnetic beads and the diluted CyPA (1 µg per 100 µg of total protein) of tris-buffered saline were flipped and incubated on a flip mixer for 1 h at room temperature. The above step was omitted in flag-tag magnetic beads. The magnetic beads were then washed three times and incubated overnight with protein sample lysis buffer at 4 °C on a rotary mixer. The next day, the magnetic beads were eluted with SDS-PAGE sampling buffer, followed by Western blot analysis.

Protein extracts were incubated overnight with specific primary antibodies and Protein A/G PLUS-Agarose beads (Santa Curz, USA). The beads were then washed with lysis buffer or PBS, and eluted with SDS-PAGE buffer. The eluted proteins were detected using WB. Flag-tagged proteins were precipitated using the Flag-tag Protein IP Assay Kit with Magnetic Beads according to the manufacturer’s instructions (Beyotime, China).