I have a protein of interest. Let's call it protein X. I treat the cell extract with calf intestinal phosphatase (from NEB). incubate at 37 degree for 1 h. Protein X would have increased mobility on SDS-PAGE gel. Does that indicate that the majority of protein X in vivo are phosphorylated? could it be due to some artifact caused by phosphatase treatment?
My protein runs around 64 kDa on SDS-PAGE gel, and the difference in migration on the gel between phosphatase-treated and untreated samples is very small. I usually run them on 10% gel. What is the recommended gel percentage to use to better visualize the difference in their mobilities?
Thank you all for answers!
If the difference in mobility is very small, it could be due to dephosphorylation. However, there is also the possibility of proteolysis. If you can arrange it, you should have an intact protein mass spectrometry measurement done of your protein. This will show whether the mass change is consistent with dephosphorylation.
It could be just a change in binding SDS.
SDS-electrophoresis works, because proteins bind on average the same amount of SDS per mass protein. If your protein has groups that disturb this average, your protein will bind less or more SDS and run slower or faster.
SDS is negatively charged, so are phosphate groups. This means they do not like each other. Therefore, a de-phosphorylated protein might bind a little bit more SDS and run a little bit faster than its phosphorylated version.
How clean is your enzyme you use for de-phosphorylation?
Are you sure it is proteinase-free or free of other enzymes that may change the protein structure?
- You could try to selectively capture phosphoprotein on columns of titanium dioxide, Ga3+ or Fe3+.
- Alternatively, you can use metal-affinity electrophoresis to identify phosphoproteins (
Article Two-Dimensional Immobilized Metal Affinity Electrophoresis f...
, doi:10.1002/elps.200800386 ). - There is also a specific staining procedure for phosphoproteins after electrophoresis (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp33300.pdf)
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