differential electrophoretic migration of phosphorylated proteins - (Sep/22/2006 )
I have some questions about the differential migration of phosphorylated versus non-phoshporylated of proteins. Non-phosphorylated, mono- and di-phosphorylated forms of myosin light chain (MLC) can be separated by alkaline glycerol-urea gel electrophoresis, the phosphorylated forms migrating faster than the non-phosphorylated one.
- Is this differential migration in such conditions specific to MLC, or is it alos observed for other proteins?
- what is the reason of this differential migration? addition of negative charges associated with the phosphate groups, or conformational changes induced by phoshporylation?
- does this differential migration persist if electrophoresis in run in similar conditions but wirh SDS?
phosphorylation means not only introducing negative charge by phosphoryls but also adding of mass (~79 Da per phosphoryl); as in SDS all polypeptides should be more or less similarly negatively masked, you mainly separate by mass; strong phosphorylation but not mono- or diphosphorylation per polypeptide would result in different migration behavior in SDS-PAGE;
there are some older discussions to similar questions in BioForum
however, in the presence of urea, charge effects are enhanced so the more negatively charged phospho-mlc runs faster in the urea gel.
in sds the difference in mw of the phospho-mlc is not significant enough to separate from mlc (sds effectively negates the charge difference).