In article <34B39B9D.61B3 at zedat.fu-berlin.de>, zeru at zedat.fu-berlin.de wrote:
>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.
Well, IMO gradients don't serve as efficient purifications steps and in this respect
are waste of time. Having said that...
>But still there are some questions open:
>1. How steep should be a glycerol gradient, if the molecular seize of
>the protein is unknown?
Should not be steep. Go by iterations here. No other way.
>2. Would it be wise to add BSA to stabilize the protein?
No (unless you protein is abandont or identitity is known). Will introduce
lots of additional contaminants.
>3. Has a saccharose gradient any advantages compared with glycerol
>gradients?
No.
>Any suggestions or comments will be greatly appreciated.
DEAE-Sephacel -> Heparin -> Mono-S sounds reasonable to try -> Mono-Q
would be my further choice/next step.
Dima
