Phosphorylation not affecting SDS-PAGE mobility - (Nov/15/2004 )

I was under the assumption that a phosphorylated protein would show a migration difference upon treatment with Claf Intestinal Phosphatase. Is it possible that a protein may be phosphorylated at sites that do not affect its mobility? What would be the reason for this?

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Yes.
OK, hypothetically....
Actually I just need to know if it is even possible to have a protein that is phosphorylated at sites that don't affect it's mobility.

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i'd wonder if CIAP can dephosphorylate protein. Pls check it up. CIAP should only dephosphorylate DNA or RNA but not protein to the best of my knowledge. you should use PPI for protein dephosphorylation

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I, myself, work on phosphorylation states of a certain protein. Phosphorylation doesn't affect SDS-PAGE mobility for a very good reason: it doesn't really change mass. I don't know the size of your protein, but adding something like 8 daltons won't change mobility at all. Also, SDS confers a strong negative charge to the protein, so the negative charges of the PO4 won't change that much.
Is it was you were wondering??

Simon

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that's about it. Great answer. So many papers rely on SDS-PAGE w/ CIP treatment to determine phosphorylation and this answers my assumption.
Thank you

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ok
thats on SDS-PAGE, how about native gels, I have seen papers that show different migration pattern for a certain phosphorylated protein, they have shown that different isoforms of the same protien when phosphorylated migrate differently, Is that right?
how do you detect those on the gel.
what about if the protien is highly phosphorylated, I mean how many phosphate groups could be added to a serine or threonin or tryrosing to make a certain protein highly phosphorylated that its migration would make a difference on the gel?
Thanks
Maggie

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I used to check phosphorylation of p70 S6K by mobility shift on SDS-PAGE and western blot using and ab with recognized both the phosphorylated and unphosphorylated protein. In my experience, mobility shifts can be seen for ser/thr heavily phosphorylated proteins in normal SDS-PAGE.

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