Hi,
I am trying to isolate 100 ug of a transcription factor in order to determine its phosphorylation sites.
I unsuccessfully tried a lot of different protocols and ended up, crude as it sounds, doing a huge immunoprecipitation under denaturing conditions.
I get one band of the correct size by silver staining, zinc-imidizole staining, and Western blotting. Hooray-except that the A260 of the HPLC-separated sample is about 60% larger than the A280. I ran an agarose gel and don't see any DNA there. Also, I can't confirm its identity by mass spec. I don't know what this band could be if it isn't my protein of interest~it doesn't match the size of anything else in my immunoprecipitation. Because it is very low-abundance and has no enzyme activity it is hard to quantify. I am wary of sending it out since I can't confirm what it is and because of the weird A260 result.
Has anyone had this experience, and what did you do?
Thank you.
K
