Phosphorylation - (Oct/11/2005 )

Hi there,

I am working on a novel protein, and I am seeing an interesting expression pattern (extra bands) in a certain disease. I think that my protein is being either phosphoylated or ubiquitinated but I am unsure. Does anybody know if there is software available to determine possible ubiquitination sites on a protein? And I am working on human tissue, is it possible to dephosphorylate my protein in some way to see if the extra bands Im seeing dissappear, or how would I determine it? Any suggestions appreciated.

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Dephosphorilate with alkaline phosphatase... if you see your bands going, there is your answer...

Or, if you think those bands are ubiquitin conjugations, use an anti-ubiquitin antibody (several available)... That should also give you an answer...

Some antibodies are also available to detect phosphorilated proteins, but I've never used any...

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you can try mass spec to determine these. But it's quite a long procedure as you need to set up a purificaion protocol that goes for your protein.
fred

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Likewise, if you exhibit dephosphorylation using a phosphatase such as CIP, it is nice to run the inhibiting reaction with sodium pyrophosphate.

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Rafflez...this is for u.
What does sodium pyrophosphate do exactly??

If (as I understand) inhibits dephosphorylation of proteins....is it a good idea to include it in my cell lysis buffer especially since i am looking for phospho-proteins like Akt, ERK.

I harvest my cells in PBS with activated sodium vanadate and then lyse them in the presence of protease inhibitor cocktail, sodium vanadate and NaF. Will addition of the pyrophospahte help??

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Sodium pryophosphate inhibits phosphatases in general....you have to be cautious when looking for phospho-proteins.
I have used the following extraction buffer when looking for phospho-proteins:

120 mM NaCl
50 mM Tris-Cl
2 mM EDTA
2 mM PMSF
10 mM NaF
10 mM b- nitrophenylphosphate
10 mM sodium pyrophosphate
10 mM b-glycerophosphate
all of the above in H20

1 ml H20 with a protease cocktail pill (Roche) ~ this is at 10X add to extraction buffer so that 1X final


EDIT - - Be sure to make this fresh each time and keep on ice

This may seem like I am being overly sensitive for the proteins....however it typically helps to maintain phosphorylation patterns.

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Rafflez....
If you use this for westerns..do u sonicate or just vortex the cells for lysis...
I work on human cancer cells

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Typically I vortex with glass beads....(20 seconds at a time for 5-6 times alternating on ice so that the samples remain cold)

Sonication works as well though....whatever your method for cell lysis you need to keep them cold

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Pria I see you are working on human cancer cell line, I'm also working on carcinoma cell line, and 'am playing around the protein lysate to see which signalling pathways are being turned on.

Well I'm using a simple SDS based lysis buffer ( which I have been using for 2 mths without preparing a new on) with the follwing composition and do pick up phosphorylated group:

20mM Hepes, pH 7.9 1M 2ml
10mM KCl 2M 0.5ml
300mM NaCl 5M 6ml
1mM EDTA (pH 8.0) 0.5M 0.2ml
1mM EGTA (pH 8.0) 0.5M 0.2ml
0.05% SDS 20% 0.25ml
dH2O 90.85ml
indicate pH 100ml

Different viwes on this topic will be highly appreciated

Lyndza

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