GST pull down assay - negative control stops working (Nov/18/2008 )
I was wondering if anyone has ever had this problem before. When I initially did my GST pull down assay, it looked beautiful- no protein came down at all in the GST alone lane, but GST +protein of interest (p.o.i.) pulled down a potential interacting protein. I repeated it with a different plant protein lysate and got another beautiful result. Then, I ran out of my GST+p.o.i. and had to make a new batch. Now suddenly, I'm getting the potential interacting protein in all lanes (GST alone as well as GST+p.o.i.). I have made new lysate buffer a number of times and I don't really see how the problem could be with the lysates. I have remade the GST+p.o.i. several times, but I'm still reusing the inital batch of GST alone that I made. I'm wondering if it's possible that something happened to the GST alone after several freeze-thaw cycles (about 6 at this point). Could it be forming some sort of aggregate that is binding to my interacting protein and potentially giving me a higher background level? I need to use a femto ECL kit in order to see the interacting protein, so a higher sensitivity to background level is possible, yet when i first did the experiment I didn't observe any background at all. I'm planning to start over with all new GST baits and lysates, buffer, etc, but I'd really like to know if anyone has ever seen this kind of thing before. How long does one normally keep the GST protein for these types of experiments?
Your input is appreciated,
smu
In my opinion, protein will degraded quickly after freezing and thawing. And you may aliquot your protein samples if you want to use it at different time.
Thanks for replying. I'm planning to do that in the future, but it still doesn't make sense to me. I've run the protein out on a gel to look at it several times and it looks fine. I suppose it's possible that it lost some of its secondary structure, but would that cause it to bind non-specifically? My boss doesn't seem to think so.