| | Re: assessing p-ERK levels after mitogenic stimulation
Thanks for your reply oBWhat
I have tried blotting for both phospho and total proteins first. The reason for trying total ERK first, is that the antibody does not bind as strongly to its target epitope as the phospho ERK does , therefore I could use a mild stripping agent (0.2N NaOH, 5 min.) to remove the total ERK antibodies. I have checked the blots after stripping this way, and have succeeded to remove the total ERK antibody completely. When I blotted for p-ERK first, I used b-mercaptoethanol(30 min. at variuos temperatures) and managed to remove the p-ERK antibodies. However, no matter which stripping agent I used, I always get the same result: the proteins in the middle of the blot is fainter than the edges. So, I tried shaking only occasionally, and the result was the same. I could try stripping less, but I guess my best bet, as you suggested, is then to run multiple blots and use different primaries. Thnaks again for your advice.