i got the two bands of 44kDa and 42kDa molecular weight of pERK1 and pERK2 respectively. Now i was wondering should we measure the two bands and compare it separately or take the ratio of pERK1/ERK2 and normalize with actin? would really appreciate your help.
It depends on what you want from the result. If you are going to tell which ERK has a higher phpspho percentage, then you'd better probe the same membrane with pan ERK antibody, which detect both phospho- and dephospho ERK proteins.
id like to measure both pERK1 and pERK2 bands. In that case, should i express it as pERK1/2 ratio or compare them individually? In our lab we dont have dephospho ERK proteins so for now just trying with phospho ERK proteins only.
Let's put it in this way. Assume you have samples A and B. For each sample, you get two bands with one Ab which detects both pERK1 and pERK2. Then you are trying to tell the change of pERK1 as well as pERK2 between sample A and B. But you can't decide how to express the phospho difference to the audiences. Did I understand your questions correctly?
Generally, EKR1 and ERK2 share 90% similarities in AA sequences between each other and have similar biological activity. We usually detected their activities together. Of course you can take the ratio of both pERK1/ERK2 but maybe proper to normalize with total ERK1/2 instead of actin.
You need to normalize both with total ERK1/2 (non-phospho) to show the fold increase in phosphorylation and then with house-keeping gene proteins like actin/tubulin etc which serves as your loading control. To your question of whether measure the intesnity of pERK1 or 2 or both, you can do either i.e shown the intensity of both bands independently or take average of both bands and express as one. Hope this helps and not too late.
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