I recently made a fresh dillution of my anti-phosphotyrosine antibody only to find that it has lost activity. It will bind to very concentrated positive controls but to a much lesser extent than before. My samples are difficult to detect to begin with so this partial loss of activity equals no signal from my samples. Has anyone had this happen? What could have caused it? Antibody is kept in the fridge and has stayed cold as far as I know. I tried new samples, new phosphatase inhibitors, new dillutions of the antibody at a few different concentrations and so far nothing has worked. Thanks for your thoughts.

yup - 'fraid so

ive had the same problem with h2b

are you sure you stored it correctly (freezing aliquots could be an option)

dom

hey,
did u see any increase in OD, when u used higher conc of Ab than before where it worked with positive control?
if there is some increase that will give u hint that Ab lost some activity (i hope your positive control is a pure protein, rather some cell lysate).
i would strongly suggest, as soon as u have your experimental samples, take an aliquot for protein estimation, to the rest add the SDS loading dye, to ensure any further loss of phophorylation event.

gud luck

We have two cases where the AB would stop working:

1. An antibody where we figured out that it only works for about 4 weeks after thawing. After 4 weeks we have to get a fresh aliqout.

2. An antibody which just got "bad" at -20°C storage. We now store the aliqout at -°80C and don't have a problem. However we can't really explain what happened.

Thanks for the responses- our positive control was a commercial stimulated lysate, and we also ran a couple of lanes of some lysate we had in the lab that we knew was phosphorylated. Why do you suggest a pure protein Dr. House? I lyse my cells and then keep the lysates on ice while I run total protein assay before I start immunoprecipitation. I run the IP overnight and then run the gel the next day. Its frustrating because I have to use all the cells I collect from a whole rat for one lane on a gel to get enough of our protein. So when the antibody freaks out like this I have basically wasted six rats.

The antibody says to store it at 4 degrees and I don't think we are supposed to aliquot it. I don't know, maybe our fridge got too warm overnight or something. If anyone else has any words of wisdom we would appreciate it!

Yes ab can stop working. Also, stick to the walls of the container, fall out of solution, etc. The conditions used to store the ab are important such as buffer, pH, and also concentration. If you need to keep at 4C you can use StableCoat for ab or StableZyme for ab enz conjugates.