Nucleotide removal from protein prep

MV. Hayes bimvh at zeus.bris.ac.uk
Thu Jul 30 06:46:23 EST 1998


Does anyone have any experience with removing tightly bound nucleotide
from protein preps. I tried DNasing. At concentrations of 2 mg/ml
DNase, the
protein (approximately 5 mg/ml) and nucleotide were getting chewed. At
lower concentrations
nothing gets removed. I have tried 3 different batches of DNase I.
I tried using polyethyleneimine (0.1-1% w/v) with 250mM salt to
precipitate the DNA but
it didn't touch it. I also tried proteamine sulphate (0.1% in the cell
lysate) incubated for 90min at 4oC.
I have dissociated the complexed protein and reassociated whilst dialysing
it against charcoal- still no joy.
I have directly added charcoal to the prep but the nucleotide hung on. 
Can anyone help. I would also be interested to know whether PEI
and proteamine remove mono or polynucleotide.
Thanks-- 

Dr Michelle Hayes
Department of Biochemistry
University of Bristol




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