degradation of signal in IP samples - (Apr/03/2012 )
I'm new to the area of IP and western blotting. I'm trying to look at the interaction between two proteins and after many attempts have been able to get the conditions to work for the IP/co-IP. I have noticed that once I run my IP samples and freeze them at -80C or -20C and when I try to reuse them to run the blots ( after boiling them again @ 100C for ~ 5min) that my signal has been going away. Both in the IP samples and the control in both IP and Co-IP situations. .
is it that my protein is getting degraded? I was wondering if there is any advice how to get around this issue.
Since I need to blot for several other proteins I can't run all gels at the same time.
did you check separation of polypeptides in gel or at least blot (f.i. Ponceau S staining)?
maybe problems with co-enriched proteases...
I have found from personal experience that over-boiling my samples can cause degradation. I used to boil my samples for 7-10 min, run them, freeze leftover, and boil again for 7 min before running them later. After doing this a few times I had varying levels of signal, even in housekeeping genes. I suspected this may have been due to degradation from boiling, so since then I have boiled my samples only once after adding SDS sample buffer, and I have not had the same issues, even when freezing leftover samples. That being said, if samples, even in SDS sample buffer, are frozen and thawed many times, I have also seen degradation happen as well.
even in the presence of sds and reducing agent, boiling samples for excessive amounts of time can cause proteins to aggregate.
if you want to use longer periods then you should use lower temperatures (eg 65 or 70C) for 10-20 minutes. this should properly denature the proteins without running the risk of aggregation.
Aliquot your samples in several tubes with SDS sample buffer. Sample from each tube is for single use.