Phosphoblot methods advice?
Claire Jessup
Friday, 03 August 2007 14:04 UTC
Is this a good place for a total signalling newbie to ask questions about blotting for phosphorylated proteins?
If so, we’re trying to look at phosphorylation of CD28 during signalling by IP with CD28 mAbs then pTyr antibody detection on Westerns. Will freezing the lysates (prior to running the gel) affect phosphorylation of the proteins. Any other tips?
If not, sorry!
Thanks
Claire
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Replies
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Hi Claire,
I think IP-ing CD28 followed by pY IB is a good start.
Freezing your samples after you finished your IP and added sample buffer at -20 is not a problem and they should be quite stable.
I have heard (not tried it myself), that IPs are more stable if you freeze them right as a pellet after you last wash. So you are only left with your beads/antibody/antigen-bearing proteins in the pellet. Then freeze at -80. Like I said, I never tried it myself, but if you are worried about stability, then I guess you could try both methods.
Also, I don’t know the molecular weight of CD28, but if it is likely to be near the heavy or light chain of your IP antibody, you should be careful to use an antibody from a different species for your IB, so you don’t block out your signal.Hope this was helpful. If there is anything else, just shout!
Nikolaus
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Hi Claire,
Welcome in our forum!
YES, you could place any comments/questions regarding signal transduction including technical questions.
I agree with Nikolaus that freezing-down IP samples does not have serious effect but I everytime observed little decrease in signal. But hardly significant.
Good lucjk!
Marek
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