I did a co-IP last week but couldn't see any bands when I developed my membrane. This is a repeat co-IP and it worked perfectly well the first time. Not sure exactly where I went wrong but I think this time around I added 300uL of lysis buffer to 15cm dish (same as last time), scraped the cells and then ended up with 600uL of cells+lysis buffer whereas last time I had around 350uL. Perhaps I haven't removed all the PBS when I washed the plates? However, I IP'd only 300uL of the lysate and stored the rest as a pre-IP.
Could it be that because I only used half my lysate that I couldn't see any bands? Can this be fixed by re-IPing my stored lysate, use a smaller vol of beads/loading buffer and then add that to my previous IP to push the protein concentration up?