I am trying to detect P-Akt in my samples but I generally observe non spesific binding. I am using cell signalling antibody (4058-dilution rate:1:5000). I have used 5 or 3% BSA for primary and secondary antibody incubation. Can anyone suggest me an established protocol or tricks that I should follow?
Hi!
I've done it for the first time this week. I used both serine and threonine P antibodies, with saturation in 5%BSA for around 45 minutes at 42 C, and a dilution of the Ab 1:1000 ON at 4c. GREAT SIGNALS! ;) I used cell singlaling as well.
p-akt(ser-473) -1:1000, and p-akt(thr-308)-1:1000, works good at 4 degree over night shaking at very slow speed. i also use CST only , washing should be perfect in btwn primary and secondary ab. secondary dilution 1:5000.
When writing these kind of questions, it is usually also relevant to tell what kind of cells are you trying to detect this in, and also what kind of lysis buffer/extraction method you re using. Such factors can have significant impact on your outcomes some times. Do you also have different conditions? (activated/freshly isolated cells?), and are you loading positive controls?
I am using mouse fibroblast cells. I have used Santa cruz RIPA lysis buffer with PMSF ,Na3VO4 and protease inhibitor. I have stored the samples at -80 to prevent degredation.
What is the stimulus you are using? As pointed out by previous responses, the phosphatase inhibitor() in your buffer is very important. But to troubleshoot and know whether it is your antibody that does not work, or you simply do not have a good detectable signal, you may want to use a positive signal you know will work. Insulin is a good choice.
Make sure you have positive control which meaning Akt in the targeting cells is activated. The procedure to prepare sample is very important, and cool conditions to lyse cells and probe Akt are better. Protease inhibitors and phosphatase inhibitors are needed. Good luck.
I agree with Peter William Krenn. And like to add to his suggestions that it also helps to serum starve cells for at least a few hours.
Hopefully the suggestions are helpful, Good luck,
Hi:
After membrane blocking 1 hour in 5% non-fat milk /TBST, We used p-Akt(ser-472) (Abcam)1:1000 at 4 degree overnight, secondary Ab dilution 1:10000. Good luck.
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