PHOSPHORYLATION - (Nov/19/2006 )

HELLO ALL
if i have a protein with a specific size say 57 , how can i tell about its size after being phosphorylated?
where to read about this stuff?
thankx

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Assuming the size is 57 KD, then phosphorylation will have little or no effect on the position of the band on an SDS PAGE gel. SDS linearizes the protein and makes the electrophoresis essentially independent of charge, and phosphorylation changes the molecular weight of such a large protein by only a very small amount.

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thankxxxxxxxxxxxxxxxxxxxx

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it depends of on the extensity of phosphorylation; about 12 mol Pi per mol protein make a shift of about 1 kDa; there are papers where scientist discriminate autophosphorylation forms of kinases by 1 to 2 kDa in appropriate SDS systems; many proteins are multiple phosphorylatable even at non-documented phosphorylation sites;

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thank u soo much

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If you run a long sds-page gel and use antibody against phosphorylated and unphosphorylated form of the protein, you should be able to see the two forms on the blot. But whenever I try to differentiate phosphorylated and unphosphorylated Rb on the western blot I am get more than two bands.

Any suggestions?

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many if not most proteins are multiple phosphorylated; in SDS only S/T and Y-phosphorylations are stable; each S/T or Y is a potential phosphorylation site despite if it is documented as a genuine phosphorylation site or not;

so multiple bands may represent different states/degrees of phosphorylation;

if you are really interested in degree of phosphorylation, try to correlate pI and phosphorylation if you know the native pI

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nowadays, i am trying to figure out how poeple assign to one protein i am working on that has molecular weight of 62KDa, and maximum of three phosphorylation sites, a 72KDa form to be its phosphorylated form. I am trying to prove that it is not its phosphorylated form simply because it can't be !!~! so up to you unphosphorylated and phosphorylated proteins (max of three sites) can;t be distinguished on simple 1D SDS-PAGE???

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yes
by using the specific antibody for ur phosphorylation sites.

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try 2D GE and determine the pI or range of pI for your protein; compare it with literature data of pI of unphosphorylated state; you may have different effects why molecular mass is increased (acyl-/glycosyl-groups e.g.); phosphorylations may contribute but only to a minor extent

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