I want to check the signal of several proteins via Western Blot (ATM, ATR, Chk1, Chk2 and the phosphorylated forms). The problem is: I don't get any signal. I tried different whole cell extracts but unfortunately without any result. The cells I'm working with are VH10tert (immortalized human fibroblasts). Does anybody have any experience with these cells? Is there anything special about preparing cell extracts for those proteins?
There is nothing special about these cells. To activate the ATM/ATR signaling pathway, one needs to treat cells with either ionizing radiations (causing double strand breaks in DNA) or DNA-damaging agents (such as etoposide). Make total extracts using RIPA buffer (see the link below) that is supplemented with protease and phsphatase inhibitors. Use 0.1% SDS in the transfer buffer to detect ATM by immunoblotting.
Link: http://www.ncbi.nlm.nih.gov/pubmed/21573174
I have faced kinda similar problem when i started looking ATM/ATR DNA repair pathway. I used gradient gel, wet transfer for 3 hrs or overnight transfer, probing with primary antibody for 12hrs at cold room and my problem solved. I agree that some time we need to activate the cell or induce the cell for injury for DNA damage response. However, which is not necessary for every cells. One final advice don't use glycerol during probing/blocking.
Thanks for your answers. It took a while, but now I got real nice signals on western blot for all proteins :) and yes, I know I have to activate the ATM/ATR pathway, so I treated my cells with BPDE
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