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PHOSPHORYLATION - (Nov/19/2006 )

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QUOTE (kosmodrom @ Jan 10 2007, 05:35 AM)
QUOTE (Kathy @ Jan 10 2007, 04:30 AM)
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. wacko.gif I am trying to prove that it is not its phosphorylated form simply because it can't be !!~! blink.gif so up to you unphosphorylated and phosphorylated proteins (max of three sites) can;t be distinguished on simple 1D SDS-PAGE???


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


on 2D this band or dots shift to the acidic part. so i suppose that should mean that this is phosphorylated form? and its mass is so much increased because of something else?

spanishflower, we dont have anti-phos antibody against this protein here.

-Kathy-

QUOTE (Kathy @ Jan 11 2007, 03:22 AM)
QUOTE (kosmodrom @ Jan 10 2007, 05:35 AM)
QUOTE (Kathy @ Jan 10 2007, 04:30 AM)
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. wacko.gif I am trying to prove that it is not its phosphorylated form simply because it can't be !!~! blink.gif so up to you unphosphorylated and phosphorylated proteins (max of three sites) can;t be distinguished on simple 1D SDS-PAGE???


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


on 2D this band or dots shift to the acidic part. so i suppose that should mean that this is phosphorylated form? and its mass is so much increased because of something else?

spanishflower, we dont have anti-phos antibody against this protein here.


it seems to be as I suggested: you likely have phosphorylation if pI/acidity increase (sulphonation may also possible); but increase by mass may predominantly happen by acylation or glycosylation; perhaps those processes are documented in literature; nevertheless you have to analyze for the precise PTM;

alternative to specific phospho-protein Ab is an anti-phosphoT, -phosphoS or phosphoY Ab; labelling with 32P-gamma-ATP is also an option; glycosylation are to detect by staining or prior deglycosylation

-The Bearer-

thank you very much for your help! smile.gif

-Kathy-

sorry i have one more question, how would glycosylated protein behave on 2D? unsure.gif

-Kathy-

1. Shift from 62 to 72 kDa in SDS-Page: We┬┤re working for years with the regulatory subunit of PKA. There a several isoforms, and none of them are running of the exact positon of the known mass (50 kDa instead of 43, 52 for 45...). And we know all the PTMs, no explanation for that, therefore we and the rest of the scientific colaboraters lump it...

2. Know I find some papers, where you see a shif of more than one kDa from just ONE phosphorylation!!!
Steinberg, 1993
Steinberg, 1996
and some more about other proteins from other groups...

Totalised there is a shift in SDS just from nothing (?) or from one P !!!

ms-olli

-ms-olli-

QUOTE (Kathy @ Jan 12 2007, 03:46 AM)
sorry i have one more question, how would glycosylated protein behave on 2D? unsure.gif


as O-/N-glycosyls are non-ionic they would not shift the pI but as there seems to be phosphorylations you may get traversal shifts in both directions of pI and mass of your protein; deglycosylation may alter the traversal to a more lateral shifting;

however, deglycosylation may also alter pI indirectly as sterically blocked phosphorylation sites by glycosyls get free for phosphorylation, or the other way round, phospho-sites become available for phosphatases after deglycosylation; all these theories may be solved by appropriate experiments

-The Bearer-

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