BioRad Rotofor

[Michael J. Studdert]

Dear Steve,
I have used the BioRad Prepcell for puriying a hydrophobic protein that I was 
unable to purify using any other chromatographic methods, even reverse phase 
HPLC.  I did not need my proteins to be conformationally intact, however, as I 
was using them to map T-cell epitopes.  The protein itself is made up in running 
buffer (reducing or non-reducing) and electrophoresed in a column of 
acrylamide gel.  I spent about 3 months getting the optimal conditions and 
found the handbook sadly lacking.  The protein is eluted from the base of the 
gel in Laemmlis running buffer with SDS in it and this was a major problem 
afterwards.  You can reduce the amount of SDS in the running buffer at least 5 
fold from that normally used but the concentration steps you need to perfom 
later just concentrate the SDS as well.  It can be reduced to very low levels 
using beads that bind SDS and exhaustive dialysis.  Our system was hooked 
up to an Econosystem with UV monitoring.  If your running smaller amounts of 
protein (<1mg) you need to run each fraction on SDS-PAGE followed by silver 
staining to see in which fractions your protein is located.  Overall it served my 
purpose but you need to consider carefully what you want to use your protein 
for once purified (it will not be in its native state) and whether conventional 
chromatography methods would work equally well if not better.  I haven't used 
the rotorfor, but hopefully these points will help with your choice.

Heidi Drummer.