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Co-Immunoprecipitation Problems with 293 Cells

Co-Immunoprecipitation Problems with 293 Cells - Immunoprecipitation Forum

Co-Immunoprecipitation Problems with 293 Cells - IP or Immunoprecipitation Forum.


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Old 01-28-2009, 04:18 AM
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Exclamation Co-Immunoprecipitation Problems with 293 Cells



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.
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Old 01-28-2009, 04:18 AM
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Default Re: Co-Immunoprecipitation Problems with 293 Cells

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.
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