Hi everyone,
I used to see my protein of interest on my blot for all my cell lines but suddenly when I rerun the experiment I don't see it anymore in one cell line! even though I still use the same antibody and I see my protein on other cell lines. I suspected that there was something wrong with the lysate so I tried lysing new cells again and it gave the same result! no bands at all!
Any ideas or suggestions on how I can revive my protein? because I have the same expression for my loading control for all cell lines so I know that there is something there >_< it's driving me mad lol
Hi Delilah,
When you say rerun the experiment, do you mean you reran the same lysates from before or did you make fresh lysates? If it is new cell lysates then I suspect the cells stopped expressing the said protein/ lost the vector. Is this an endogenous protein? I don't think it is a western problem as you are able to get the protein in other cell lines but if you still want to check it, I would just do a quick dot blot (as in just put a ul of your lysate on the membrane, let it dry and probe it with antibodies) to see if your protein is there in the lysate.
Certain proteins are affected if its extract/lysate had to undergo multiple times of freeze-thawing. The proteins are either denatured or its structure got further cleaved due to the harsh effect of freeze-thawing, especially the unstable proteins. For example, I have experienced either lower or no expression of vWF protein when the same extract batch was freeze-thawed for the second time. Therefore, I applied one extract for only one time of western blot run. One suggestion will be to store your lysates in aliquots so that you don't freeze-thaw your proteins many times.
Many thanks Sajjeev Jagannathan , Yes I used a fresh lysate so most likely the cells are the problem. it is an indigenous protein I have not transfected the cells or anything.
Thank you again, I think I'll give the dot blot a shot!
Because your antibody is not of that quality you need.
See: https://www.nature.com/news/reproducibility-crisis-blame-it-on-the-antibodies-1.17586
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