Hi Daniel: If only life were that easy. RUBISCO is a difficult protein to
get rid of completely in the inital stages, also most plant extracts have a
lot of RUBISCO degradation products, which light up as background with many
antibody preps, since the host animals have varying amounts of IgG to
RUBISCO. We have had some trouble with even secondary antibodies (esp
rabbit) binding to RUBISCO.
The classical means was to precipitate it with Mg2+ at near its pI (I think
HEPES, 6.8 and increasing Mg). Another possibility is to perform
centrifugation of carefully extracted plant materials on a sucrose
gradient, RUBISCO (most of it will sediment away. I will try to locate
some refs for you in the next day or so. Another way to fractionate with
ammonium sulfate, most of the RUBISC protein will fall out at 60 %
saturation. Howvere, a bulk of the cellular proteins will also precipitate
at this salt oncentration. oThers have used PEG pption,, but in my hands
this has had mixed results. It alsodepends on the amount that you need to
get rid of. If working with cytoplasmic exts, you can try to spin out the
chloroplasts, but RUBISCO contamination is fairly high.
You should be able to get antibodies to RUBISCO from several researchers.
Immobilized anti-RUBISCO work to different extents, depending on the titer.
I will dig out some more info if you need it, good luck, gautam
Gautam Sarath
N-226, The Beadle Center, 19th and Vine Street
University of Nebraska, Lincoln
Lincoln, NE 68588-0664
Tel: 1-402-472-2928; Fax: 1-402-472-7842
http://www.biotech.unl.edu/Proteins/index.html
http://www.pigment.unl.edu/dept/sarath/sarath.html
This archive was generated by hypermail 2b29 : Fri Feb 23 2001 - 13:03:33 EST