Concentrating enzymes--more details
arsphys at CC.USU.EDU
Wed May 26 11:27:28 EST 1999
Thanks for the suggestions so far. I didn't want to make my
first request too long,
and I guess I omited a few too many details.
My procedure is:
1. Grind tissue in 4 liter Waring blender in acetate buffer w/
PMSF, EDTA, benzamidine, EACA, DIECA, ascorbate, & PVPP.
2. 45-70% Ammonium Sulfate Cut
3. Desalt on BioGel P-6 in 5 mM acetate with above additives
plus 5% glycerol and minus PVPP. (Column desalting gives me
back twice the activity as dialysis--I think the enzyme binds to
the dialysis tubing--that's why I have avoided PEG in dialysis
It is at this point that I need to concentrate 4-5 batches (200
ml) to 45-50 ml prior to preparative IEF (Rainin). ConA
Sepharose follows that.
I have tried at later stages: Centricon, Centriprep and
Centriplus devices (although all these are cumbersome for 200 ml
samples) and I frequently lose activity with them also. I think
this is due to inactivation since activity decreases relative to
protein conc. (specific activity goes down).
I am currently looking into freeze drying with column desalting.
Any other suggestions for concentrating?
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