I've begun examining ERK phosphorylation in vivo and previously my antibody for total ERK (Sigma M5670) gives me 2 bands (ERK1/2) in my HEK293 cell line, but now using it for mouse brain samples I'm getting three bands, the third band showing up at around 46 kDa. I have read that there is an isoform, ERK1b,(Yung, Y., Yao, Z., Hanoch, T., & Seger, R. (2000). ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK. Journal of Biological Chemistry, 275(21), 15799-15808.) that is 46 kDa, and wondering if this is the band that I'm seeing and if this is normal.
Thanks,
Tom
Tom,
Your thought is probably right. The newly pre=print in F1000 has an article recalculate mouse ENCODE data found transcriptome is conserved in tissues cross species. HEK293 is from kidney, and we know brain has the most diversified alternative splicing comparing to other tissues. If you have mouse kidney tissue as well, you can use it as a comparison in WB. Meanwhile, you can perform RT-PCR to see if there is a match isoform in RNA level.
hey thomas !
Though i recently started facing the same problem but
as far as i know phosphorylation does not bring any significant change in molecular weight of p42/p44 mapk
the position of the band essentially remains the same in SDS page even after phosphorylation
Check the link below to see how post translational modifications brings about changes in molecular weight
http://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/phosphorylation/mass-changes.html
Please let me know too, if could get a solution to this !
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