phosphorylation analysis - (May/04/2006 )

Hi all - I've been working with a putative transcription factor. On WB's I've been getting 2 bands, separated by approx 5,000. the question has come up -is the protein being phosphorylated? Models do predict a possible phosphorylaion site w/ threonine in the pocket. Any ideas on the best way to analyze. I've been thinking about using an anti-phosphothreonine but maybe there is a better way to go about this.

Thx!

Hi
A friend of mine did a lot of work looking at phoshorylation of his protein...I think a simple test might be to treat your protein with phosphatase to see if the size changes. Check out this paper (and related ones), there might be some good ideas for you:
http://www.pubmedcentral.gov/articlerender...bmedid=11602713

Cheers!