Help needed: glycerol gradient centrifugation

Karin Zerulla zeru at zedat.fu-berlin.de
Wed Jan 7 10:13:33 EST 1998


Dear netters,

I'm trying to purify an unknown DNA-binding protein complex using
biochemical methods. The DNA-binding activity is detectable after
DEAE-Sephacel-column and Heparin-Agarose-column purification steps, but
it's lost while glycerol gradient centrifugation.
In the meantime I've learned that my purified protein extract was
probably too diluted and I should better use a vertical rotor instead of
a swinging out bucket to shorten the centrifugation time.
But still there are some questions open:
1. How steep should be a glycerol gradient, if the molecular seize of
the protein is unknown?
2. Would it be wise to add BSA to stabilize the protein?
3. Has a saccharose gradient any advantages compared with glycerol
gradients?

Any suggestions or comments will be greatly appreciated.

Thanks in advance

Karin Zerulla
E-mail: zeru at zedat.fu-berlin.de



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