Hello,

I am trying to conduct an IP between two proteins (protein A- 97kd and protein B-37kD). For my experiment, I am using 0.5X RIPA buffer with the addition of protease and phosphatase inhibitors. I am using ~1mg of protein lysate for 1ug of antibody and incubating the samples overnight. The next day, the samples are incubated with beads between 2-4 hours. For washing the beads, I am still using the same 0.5X RIPA buffer. When running the western blot, the proteins that I used to pulldown are detected however, the protein that it is in complex with is not detected. I am unsure of why/how the protein of interest is being pulled down, but its complex members are not.

Any advice will help! Thank you