Trouble in phosphoprotein detection - (Feb/28/2006 )

Hi, I m trying to detect cell signalling activation states in NIH ovcar 3 cells, and clones expressing a particular epitope using complete medium and serum free. The molecules I m trying to detect are AKT, EGFR, MAPK, GSK 3B (and their phospho `s).
Although all the antibodies I use are fresh, my results are really inconsistent. I m using a proteases - phosphatases cocktail that includes leupeptin, apoprotein, AEBSF, PEPSATIN, NAF, Sodium orthovanadate, s. pyrophosphate. But I do not know if these are the best for such set of phosphoproteins.
Can you recommend me any tip to detect these molecules. Thank you so much!! :blink:

Hi, I m trying to detect cell signalling activation states in NIH ovcar 3 cells, and clones expressing a particular epitope using complete medium and serum free. The molecules I m trying to detect are AKT, EGFR, MAPK, GSK 3B (and their phospho `s).
Although all the antibodies I use are fresh, my results are really inconsistent. I m using a proteases - phosphatases cocktail that includes leupeptin, apoprotein, AEBSF, PEPSATIN, NAF, Sodium orthovanadate, s. pyrophosphate. But I do not know if these are the best for such set of phosphoproteins.
Can you recommend me any tip to detect these molecules. Thank you so much!!

the buffer seems to me Okay, too.
cells are sensitive to centrifugation, vortex...it can activate them.

I've been using some of Cell Signalling's antibodies to detect phosphorylated proteins [MEKK, JNK, p53 amongst others]. Some other guys in the lab were doing the same. I was getting really good results and they were getting inconsistent results [though they always had to expose their blots for way longer than me to detect their phospho-proteins], so we went over how we each do our Western blotting [we were all following the exact protocols for these antibodies as supplied on the antibody datasheets].

Turned out the only difference in the way we were doing things was the membrane we used - they were using Hybond [Amersham] and I was using Protran [S&S], which are both nitrocellulose so you'd think there shouldn't be any difference. But, when they switched to using Protran they started getting great results. Also, during a recent Cell Signalling exhibition I asked the rep about this, and he confirmed that for anti-phospho antibodies [their anti-phospho antibodies anyway, you don't say who you're getting yours from] sometimes even things you think shouldn't matter - like the membrane you use - can make a big difference.

So: You could try another membrane, just in case it makes a difference?

The Cell signalling Phospho STAT1&3 and Phospho ERK abs work well in our lab using Hybond ECL NC membrane (Amersham). We use TBS-0.2% Tween 20 as a wash buffer and always block and dilute the phospho-antibodies with 5% BSA/TBS-Tween 20. Milk is supposed to have a lot of phospho proteins in it so I've heard so could affect your results. I've also read on this forum using PBS that phosphate groups could interfer with alkaline phosphatase detection (but also with phospho abs? not sure if that's right though).

All the best,
Ceri