My enzyme is of low abundance in leaves and it runs slighly below rubiscoLS
on SDS-PAGE gels. This cuases a problem with immunoblotting. When to much
leaf protein is loaded rubiscoLS seems to cover-up or mask or distort my
enzyme band so that it is no longer bound by the antibody. I can only
detect my enzyme by loading small amounts of protein (2 =B5g total protein o=
mini gel system), but the signal is weak and difficult to photograph for
illustrations. I would appreciate any advice to improve the immunoblot
(1) an easy technique for selectively removing rubisco- does anyone have a
good antibody against rubiscoLS that can be used to immunoprecipitate it?
Has anyone tried affinity purification of rubisco on an RUBP-Agarose resin?
Is there a simple treatement like heating that will denature rubisco?
(2) a gel system that will better resolve proteins in the 50 kDa range
or other ideas.
Thomas Leustek, Ph.D.
Center for Agricultural Molecular Biology
59 Dudley Road
New Brunswick, New Jersey 08901-8520
Telephone: (732)932-8165 ext. 326
Email: leustek at aesop.rutgers.edu