assessing p-ERK levels after mitogenic stimulation

[mickey07]

Hi All

I want to look at the level of phosphorylation of p-ERK (p44 MAPK) after mitogenic stimulation. I am using the p-ERK and total ERK antibodies from Cell Signaling. My problem is that I wish to use the same membrane to assess the levels of both these proteins, but I am experiencing difficulties with stripping membranes (loosing proteins) and not getting good signal afterwards. I have tried different methods of stripping as well alternating these antibodies before stripping , but to no avail. I know that to assess the phospho levels of ERK, conventionally, one need to probe for both p-ERK and total ERK and then determine the p-ERK/t-ERK. My question is whether it would be acceptible to determine the level of phosphorylation in my mitogen treated samples compared to my control, which is untreated sample (t0) and normalising to a housekeeping protein, eg, GAPDH, rather than using the p-ERK/T-ERK ratio. Thanks in advance for any advice.

[oBWhat]

Hello,

are you stripping your blots after you blot for phospho-proteins? that would be your best bet, blot for phospho-proteins and then strip the membrane to blot for total protein.

If you are doing that, or need to strip more then you need to load more protein or strip less.
Either that, or you can do multiple blots with more than 1 primary.
I have done that before.

Anyone else care to comment?

[mickey07]

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.
Ciao