Perhaps also protease inhibitors are needed - e.g. (from Santa Cruz) Complete™ Protease Inhibitor Cocktail Tablet, EDTA-free, Cat. No. sc-29131.

Sounds like your sample target protein may have had Ab-specific epitope degradation slowly over time by some kind of protease?

There are also protease inhibitors and plus the total protein is not degraded...

Hi, Gilda, not frustration, this is not help! have you stained both membrane with PONCEAU? Do you have any protein on the mebrane after treatment with phosphatase inhibitors? If the treatment with phosphatase inhibitor was lead to total protein degradation, you have to order new one. Have you tried to make detection of the any other proteins in the mebrane after tretament with phospahate inhibitors?

This is one of the importnat control!, Good luck!

That is a very tricky situation then, indeed. Slow spontaneous dephosphorylation or another unknown reason for it is hard to fathom as well. Stumped too.

I think there is some technical prob, once i have prob like this, i have tried few tips, slow running on gel, increased concentration of protein and use of fresh antibody, and if u are using cutting stirps of membrane for the target protein then possibility of missing of the target atrea so increase the width of membrane strip, but i achieved my target after alot of struggle

I stained both memebrane with ponceau, the proteins are there and after staining for my antibody and getting no signal I stain for H4 and get a perfect signal. there is no dedgradation going on as the total protein is perfect on the other membrane. by the way i stain the whole membrane and do not cut the strip. I do not understand I just do everything as I always did and it always worked...

Hi Gilda, all the research known the frustration of "I just do everything as I always did and it always worked", a time in their life!

I think that the problems could be :

ANTIBODY

SAMPLES

You said you had no probelms in the past but you made the same experiments with the same samples? For examples I stimulated my cells and than studied phosphorylated and total p38, what you do this time, stimulated cells in the same way? Or you used tissues samples? Somenthing changed?

Can you find positive control, for example you still had some old sample that work in the past, in which you detected your phosphorylated isoform? First you have to understand if the problem is the antibody, if the antibody work well ( for example cell signaling antibody are excellent also recycled five times!) you have to understand if something changes the phosphorylation state of your isoform.

You activated other pathway in which are involved other phosphorylated protein? You can check if you can detect those phosphorylated proteins to understand if the ihnibitors work well.

I do not known you work in details so I cannot think more suggestions than this, sorry.

Are you certain that the phosphatase inhibitor is working as advertised?

can the sample buffer be a problem if the formulation is wrong? i mean can it leave the total protein run good but make me lose the signal for the phosphorylated?

because now I see some bands but the background is too high, if I wash more I lose the signal, if I put the secondary again no signal as the primary has been washed away too. So can it be my sample buffer that makes me lose the phosphorylation?

...senescence of any/all reagents might be suspect here - especially if others are using (and accidentally abusing) the reagents you are using ... 'expired' DTT, DTE half-life tolerance may be a factor in the sample buffer if C-C crosslinking has occurred on account thereof ...?

if you are detecting a phosphorylated protein, try to use BSA INSTEAD OF non-fat milk for bloking and 1st Ab dilution; while performing 1st Ab binding, do 4 celsius degree overnight. (these are suggestions from someone I knew and who obtained beautiful WB results). CHEER UP!

thank you Menagro, this is what I already do and my westerns were just perfect...I hope I can solve this problem soon...

Hi. I have experienced the same problem. Usually, is the primary antibody, that for some reason (mainly human mistakes) is contaminated, diluted, someone forgot to keep it in the freezer again...so Is not working anymore. I would ask for an aliquot or buy it again.

Other problem I experienced happened when samples I received where homogenated in laemly buffer with Beta-mercaptoethanol and that boiled several times. In this case signal quality was gradually deteriorating.

However I would first of all try with new antobody.

Hope It will work.