unstable flag tagged protein - (Dec/22/2011 )

Hi
I used the expression vector pCDNA3.1/flag to subcloning the C-terminal region of BRCA2 and transfect it into BRCA2 deficient cells. After Immunoprecipitation and Western blotting analysis with a flag antibodies, I didn't see the expected band of approximately 80 KD but two bands of 50 and 30 KD.
Any suggestions?
Thanks in advance
Laura

did you run the Antibody along with the IP, just to make sure your 50KD is not the Ab chain.....

No, I didn't! Thank you for your suggestion, I'll verify it.
Just an information, how many antibodies I have to run?

i usually denature the same amount i use for IP.....may be u can also use bit less than that...

Ok!
Thank you very much!