Would IP make the protein of interest run slower on SDS-PAGE? - (May/21/2013 )
Hi, I have done an IP with a rabbit antibody. I then resolve the sample by SDS-PAGE and immunoblot again with this rabbit antibody, then an anti-rabbit secondary.This is fine as my protein of interest is around 75kD, so the heavy and light chains dont interfere with my protein of interest band. However, the IP protein is higher than usual, and i was wondering whether this could be due to the IP/protein complex, would it run slower because of the IP?
Studies have been done on this protein before in which a higher band is present but this was full lysate after transfection, not endogenous IP.
The protein is also glutamine at the C terminus which I read could make the sample run slower on SDS-PAGE and resolve at a higher mW? Also, the higher protein is not post transcriptionally modified as work has been done to see why this higher band appears and the two species are the same. It was sort of decided that the polyglutamine region was why this occurs.
I'm unsure of which is right and I need to know as I am sure it will come up when I give a talk on my work.
Many thanks guys
I have run a lot of IPs and never had this problem, but I've heard people say the band is at a higher weight than normal. This should not happen however. You must check with controls.
have you determined the mobility of the protein in sds-page prior to ip?
can you use a different technique to determine the size of your iped protein?
sds-page is not an absolute method for size determination. there are a number of factors which may affect the apparent size.