Posted 02 February 2011 - 05:51 AM
I hope I'm in the right place to pose this question... I'm working with a transcription factor that we think needs to be activated by phosphorylation to form the active dimer. My question is, how do I phosphorylate my protein? It might be helpful to know that I've had a problem with keeping my protein soluble in the past (it's currently soluble now in 20% glycerol, 50mM Tris-HCl and 250mM NaCl) and that my end goal here is to study the protein-DNA interaction.
thanks in advance for any help!
Posted 02 February 2011 - 08:22 AM
Posted 02 February 2011 - 09:26 AM
Thank you so much for your insight. As a masters student I'm finding that a lot of work is "trial and error" ...
My target protein is part of a two-component regulatory system, so I was thinking of using the kinase that proposedly phosphorylates it in vivo but, seeing as it's a membrane bound protein, wouldn't co-expression be somewhat less efficient if I express them in E.coli BL21 cells? Also, with point mutations, seeing as my protein is unstable, wouldn't this additional negative charge cause it to precipitate more easily?