Two bands in WB but one band in IP - (Apr/15/2015 )
Hi,
My protein of interest is around 70 KD and I have seen there is another isoform at 100 kD. When I do western I can see both the 70 and 100 kd band. However, in my Immunprecipitation I could never see the 100 KD band , only the 70 KD band pulls down with my IP. This I have seen more than 20 times , so its reproducible.
I was wondering, what might be the possible explanation for it. Why the 100 KD disappears in IP ? any good scientific explanation will be highly appreciated.
Thanks.
Hello,
Interesting observation. Do you observe this phenomenon with two separate antibodies, or one antibody used for both IP and WB? Polyclonal or monoclonal, respectively?
This could be for several (speculative) reasons:
- Are you certain this 100kDa form is the same protein as the 70kDa protein? Is this larger isoform actually an alternative isoform (predicted by mRNA isoforms)? Or alternatively, is this the same protein +/- modifications or proteolytic cleavage?
- It's possible that you simply have a non-specific band in your western blot. Generally IP Westerns "clean up" a dirty western blot from lysate by removing such non-specific banding patterns, so you may simply be removing noise by doing the IP-Western. This would indicate that the 70kDa band is really your protein of interest.
- It is possible that the IP antibody only recognizes an epitope that is revealed by post-translational processing or produced by a tertiary structure present in the 70kDa form.
- Similar to the previous possibility, the 100kDa isoform may contain post-translational modifications (acetylation, hyper-phosphorylation) that change its antigenicity with respect to your IP antibody.
- The 100kDa form might be in complex with other proteins and hidden from the IP antibody.
There are a lot of possible reasons, but without knowing the biology of the protein itself, it will be difficult to guess. What do you think might be the cause, given what you know of about the protein?
Cheers