Co-Immunoprecipitation Problems with 293 Cells

[clokke76]

I transfected 293 cell with my protein-V5/protein-FLAG plasmid, protein-V5/FLAG-Vector, protein-FLAG/V5-vector, and used 293 not-treated as negative control, to detect whether the protein form dimer or not. I used anti-M2(FLAG) to do IP and used anti-flag or anti-v5 to do IB.
But every sample have a bond about 90KD and the size of my protein is about 90KD!!!
I have repeated times and changed to use CHO-K1 cell, but the results were similar.
It drives me crazy,,, could someone help me out?

By the way, the V5 vector is pCDNA3.1 V5-His B and the FLAG-vector is pFLAG-CMV-1, all the constructor have been sequenced.

[kiki06]

hey there,
some proteins bind specifically to IgG, for example the protein actin.


IgG molecules are "sticky" by nature more so than most proteins in my experience. I have seen the same results you have, try increasing the salt concentration, or the pH in your wash buffer. In one case I saw someone with a wash buffer that had a pH of 9, yet still retained w/their coIP complex.

Remember, a co-IP DOES NOT mean you have a protein-protein interaction. It means that your two proteins are found in the same complex.