I am recently performing Western Blot for a protein.
After treatment, the apparent electrophoresis mobility increases detected by its phosphorylation antibody. Moreover, the other protein molecular weight increases detected by its total antibody. So, does anyone know what had happened for such proteins? Are they modified post-translationally? And which modification will change the molecular weight of a protein?
Any good advice about such modification for further study will be helpful!
Thanks a lot in advance!
Zhiyuan
A phosphorylation can give a small shift to the protein as compared to the non-phosphorylated form.
It is a bit hard to say from your question, but if you are going from a calculated MW to the gel MW a small shift either way can be expected. Ladders are not perfect and if your protein has a charge this will effect how it runs on a gel. Once you have an expected weight you can then look for changes after treatment. This can be cause by changes in the phosphorylation or glycolosation or ubiquitinisation. The direction of movement will be related to both the change in the weight and the change in the charge.
Hi Sandra,
Thanks for your reply immediately. Your comment about charge change for protein is perfect. And various modification also exsits. Further, what will you do about the MW shift a protein?
Thanks
Zhiyuan
Please check this link for acetylated protein gel shift:
http://www.nature.com/nprot/journal/v5/n9/fig_tab/nprot.2010.117_F2.html
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