hi everyone, I am getting crazy.
Last year I did a lot of WB with very good success. I used a set of 3 antibodies and it worked very well. My developed blot showed to unspecific bands or even a Background. I got exactly the bands I supposed to see; a single band each. I incubate primary Ab over night in a fridge.
Then suddenly from a day to another I've got high background and band laddering. I tried several time using new buffers, new antibody solutions and everything new. Always the same. Then a colleague did for me and it worked again. We did some changes like removing sodium acide from the Ab solution.
Today I did again by myself and the WB failed again. For me it looks like my antibody is denatured over night and binds to every protein that was transferred. The overall background is acceptable low.
So my question is, what may let an antibody degrade, even after preparing a fresh solution. Is it possible to denature an Ab by SDS or protease? And would this cause a laddering effect?
many thanks for any reply