I have recently been doing IPs on two proteins we hypothesize interact. All of our controls are showing what we completely expect, suggesting antibodies are specific. When we do an IP, we get the bands we expected, but they are running at a lower MW than the combined predicted weight of the two interacting proteins. Has anyone else seen this and what might be the explanation?
Hi, @Richard! At what conditions do you run electrophoretic separation of protein bands? If you use denaturing conditions with SDS the non-covalent protein-protein complex will be most probably destroyed. You should verify the observed MWs with the predicted MW of individual proteins.
I am not sure, if I correctly understand the question. Is this a native PAGE gel that you are using, or did you crosslink the interacting proteins prior to the IP? If not, the SDS in the sample buffer (and the heat typically applied for SDS loading) will denature and disrupt your protein complex.
Aside from that, there are many reasons why proteins may run at an unexpected size. These, for instance, include post-translational modifications including phosphorylation or glycosylation. The size aberration could also be an experimental artifact caused by a pre-stained marker. I have seen pre-stained markers consistently run aberrantly when used on large SDS PAGE gels (25-30 cm), which were run very slowly overnight. In fact, we had to re-calibrate the marker for these runs. The problem did not appear on conventional small SDS PAGE gels run within 1-2 hours.
Another stupid question: Are you sure you are detecting your co-precipitated proteins in the gel and not merely your antibody used for IP?
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies