I am a student of Dr Gary Small's at the University of
South Dakota. I am trying to resolve our proteins (blue light
photoreceptors) on a 2-dimensional gel and was wondering if
anyone else has attempted 2D gels. I am able to solublize my proteins
and am having some luck with the focusing and subsequent SDS-PAGE.
Although I am using a simple 1st-step for solubilization (suspension in
40mM Tris Base followed by brief sonication and centrifugation), the
majority of the cellular proteins are in the supernatant. I believe I
will be able to optimize the focusing step if I can further "purify"
this soluble portion. Does anyone have any suggestions for doing step-
wise solubilizations of Chlamy extracts? Urea and any non-ionic
detergents are compatible with the focusing step but salts are not.
Thank you in advance for your help.
nvavra at usd.edu