Karin Zerulla 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.
If you are doing rate zonal centrifugation (separation by size) you
should stick with swinging bucket or vertical tube rotors. Fixed angle
rotors can be used for isopycnic centrifugation (separation by density),
because this is an equilibrium technique and wall effects are not
important. Note that there are quite small swinging bucket rotors
available, if necessary on a desktop ultracentrifuge. Remember that for
rate zonal experiments, the loading zone should be no more than 1-2% of
the gradient, therefore use fairly concentrated samples. Also make sure
that the density of your sample is less than the start of your gradient!
> But still there are some questions open:
> 1. How steep should be a glycerol gradient, if the molecular seize of
> the protein is unknown?
Usually something like 5-30% is used, but that depends the rotor
geometry. The idea is to create a isokinetic gradient, where the
increased centrifugal force experienced by the protein molecules as it
moves outward is just offset by the increase in density of the medium,
so that all particles move with constant (but size dependent) speed
through the gradient. See the paper by Martin and Ames (JBC, some 30
years ago) for details. You then adjust for the molecular weight of your
protein by changing the run time.
> 2. Would it be wise to add BSA to stabilize the protein?
No. You create more problems than you solve (contamination, gradient
inversion).
> 3. Has a saccharose gradient any advantages compared with glycerol
> gradients?
It is cheaper. Other than that, it depends on the idiosyncrasies of your
protein. Another alternative are iodinated gradient media like
metrizamide. With those you can create isoosmotic gradients, which may
be important if your protein does not like the high osmotic pressure in
glycerol or sucrose gradients. They are very expensive, however.
