STAT-3/phosphoSTAT-3 western blot - (Mar/14/2007 )
Hi everybody,
I would like to perform an experiment to assess the amount of STAT3 phosphorilation upon stimulus in mice dendritic cells. I was thinking of doing a western blot for STAT-3 and pSTAT-3, using Santa Cruz antibodies. Do I have to perform two separate blots to identify the two forms (phosphorylated and not phosphorylated)? And if so, how can I quantitate the phosporilation?
thank you
Margherita
You can do this blot very nicely, I have done it. But please use P-STAT3 antibody from Cell signaling, saves you a lot of trouble. There are two different phospho-antibody, for serine or tyrosine phosphorylation of STAT3. Normally, you first look Tyr705, that would be antibody 9138. For normal STAT3 you can use santa cruz antibody sc-482, this one is very nice working.
You first do the blot for P-STAT3, strip the membrane, probe against STAT3, strip again and probe against GAPDH or Actin. Thats the nicest way to do. I would use PVDF-membrane for this. And strip with low pH instead of ß-ME, but that's just my favorite stripping method, no bad smell.
Hope, this helps for beginning.
You first do the blot for P-STAT3, strip the membrane, probe against STAT3, strip again and probe against GAPDH or Actin. Thats the nicest way to do. I would use PVDF-membrane for this. And strip with low pH instead of ß-ME, but that's just my favorite stripping method, no bad smell.
Hope, this helps for beginning.
I suggest to do first the anti non-phospho STAT3 Ab, strip, and then anti-phospho STAT3 because the anti-phospho Ab may give the stronger signal which is more difficult to strip - but if you expect no or a low phospho signal, do it as biomaus suggests
My experience is the opposite, the signal of P-STAT is mostly not so strong, STAT is constitutively expressed in most cells and the antibody detects very good the protein. But just give it a try as you like 
Thank you guys, I think I will first try with biomaus protocol. By the way, can you explain how to strip the membrane with low pH? We usually use b-mercaptoethanol.
Sure, you just wash your membrane 3 times in TBS-T for 10 minutes, than put it in a box in 70°C water bath with this stripping buffer (Stripping buffer: 0.2 M Glycine (pH from 2,5 to1,8, as you like) and 0.05% Tween 20, keep solution at 4°C) on it for 30 minutes, than wash again 3 times with TBS-T for 10 minutes, than start again with blocking.
This is a little bit milder stripping than with ß-ME. You can also have a look an the abcam-site, there they have this protocoll also:
http://www.abcam.com/ps/pdf/protocols/Stri...20reprobing.pdf
thank you,
as soon as I have the reagents I will go for it