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phosphotyrosine blot


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#1 max muster

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Posted 29 April 2009 - 08:57 AM

Hi,

I hope someone can help me out with this:
I am working with human dendritic cells, which are supposed to show increased protein phophorylation after stimluation with LPS.
But in my western blots with phosphotyrosine-specific antibody I don't see any protein phophorylation compared to the unstimulated control.
I have tried different LPS concentrations and incubation times, but have had no success so far.
So I'm thinking there might be something wrong with my protocol:
The cells are grown in a 3ml well. after stimulation with LPS for 15 min, the wells are put on ice for 20min.
The cells are then washed with ice-cold PBS two times, the pellet is resuspended in RIPA-buffer + PMSF,aprotinin,leupeptin,pepstatin,Sodium fluoride and sodium orthovanadat and kept on ice for 60min. The tubes are vortexed and pipeted up and down every 15 min.
After centrifugation I take the supernatant and use it for a SDS-PAGE- westernblot.
Any ideas why there's no phosphorylation visible? I've also tried adding the phosphatase-inhibitors during the antibody incubation/washing procedures, which didn't help.

#2 mikew

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Posted 30 April 2009 - 01:55 PM

Hi Max,

I have no experience with dendritic cells.
However, I have treated various other cell
types with various stimuli that is supposed
to induce phosphorylation and have never seen
a change on blots from whole cell lysates.
I only see changes when I immunoprecipitate
specific proteins and look for specific changes.
The reason for this is that in a typical cell 66% of the proteins are already phosphorylated, if you induce with
LPS or whatever, and this induces hyperphosphorylation of 100 proteins this is still a small fraction of the total.
Also, you may not notice hyper phosphorylayion versus basal phosphorylation on a whole cell lysate basis.
Do you have a reference for where people have seen this sort of change?
Also, check to make sure your LPS treatment is working.
For example, is NF-kB activated or some other control?

#3 max muster

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Posted 03 May 2009 - 11:10 AM

there are several publications out there, where a change in phosphorylation has been observed, for instance:
http://intimm.oxford...full/16/12/1701
although i have to admit, i've never seen a paper with an unspecific phospho-blot.
we are using the unspecific phosphotyrosine-blot as a kind of "screening"-method, to see which proteins are being phosphorylated after exposure to LPS. with that knowledge, we would then like to blot with a specific antibody for these proteins.
do you think this could be the problem and that we have to use a specific antibody right away?
The LPS is working fine, I have a good response in my ELISA and NFkB is activated in my nuclear extracts.

#4 mikew

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Posted 04 May 2009 - 11:21 AM

Hi,

Yep, I bet the problem is that you are using a pan-phosphotyrosine antibody.
If you use a phospho-specific one I bet your experiments will work.
Glad to hear you have a control for your LPS activity, that always a good idea.
I have used pan-phopho-tyrosine, serine and acetylation antibodies and haven't seen changes on a global scale.




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