having problems with p-erk antibody and other phosphoproteins antibodies - (Jan/17/2014 )
My westerns have been fine with regular antibody (non-phospho antibodies). However, with phospho antibodies, the results have been really inconsistent, and sometimes even inverse of someone else's (using the same cells from the same flasks). I do use phosphatase inhibitors and process everything really fast/on ice (to prevent phospho degradation). I can't figure out what else might be the concern since everything else has been the same (using the same reagents and techniques as someone else in the lab but not having the same results). Is there something that I might be overlooking?
Thanks so much in advance.
edit: I do get bands for phosphoproteins but they're not the results I'm looking for (i.e. compared with someone else who used the same cells from the same flask).
have you compared your protocols with those of your lab mate (including timing and handling)?