[Protein-analysis] question

Kate Chabarek kchabare at cats.ucsc.edu
Thu Jul 13 18:32:24 EST 2006

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.

More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net