I'm looking for an intra cellular protein phosphorylation through Western Blotting, as I'm following a protocol specific for phosphorylated protein, I can not use GAPDH and b-Actin as housekeeping genes, so I need a phosphorylated protein which I can use as a housekeeping gene.
Actually the question is not so simple to answer.
fist of all consider the tissue (or cells) that you are using, normal vs. diseases. Also consider the experimental setting, it may affect the expression of the housekeeping gene and thus the amount of protein.
Than you can run a simple WB for and probe for GAPDH, b-ACTIN and b-TUBULIN and see if there are some differences. For the phosphorilated protein just normalize for the dephosphorilated version.
for housekeeping gene WB comparison I suggest to read
- Ferguson RE et al Proteomics. 2005 Feb;5(2):566-71
hope it can help
When you say you are using a protocol specific to phosphorylated proteins, does it mean that you are enriching your protein extractions for phosphoproteins and actin/GAPDH are thus absent from your samples?
I do not understand the question.
Why is it necessary for you to normalize to a phosphorylated protein? Please explain!
As said above you can use any unchanging protein for normalisation which proteins are not changing depends on the tissue and treatment choice and there is extensive literature on that question. If you look for a constitutively phosphorylated protein, there are some, but I am not shure, if there are specific antibodies against these.
Thank you guys for your kind reponses!
To explain my question, I'm looking for STAT-5 and JAK-2 protein phosphorylation after treating the cultured Fibroblasts with apoptosis/anti-apoptosis substances.
According to the difference between the Phospho-protein and unphorpho-protein protocols for western Blotting, specially the Blocking step!, is it possible to use the b-Actin or GAPDH antibodies as housekeeping gene on the same membrane?!
If you want to normalize p-JAK-2/GAPDH and JAK-2/GAPDH, then, mathematically, GAPDH disappears; so no need to normalize for an housekeeping gene.
If you are using a gradient gel (let's say 4-12%) you can cut the membrane in different parts. Thus you can run multiple western blots.
My suggestion is to incubate the p-Protein with BSA and the un-phosphorilated protein with milk.
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