elarson at ux1.cso.uiuc.edu
Sat Sep 10 01:13:03 EST 1994
jbooth at ACS.UCALGARY.CA (Joseph Booth) writes:
> I've read that you can concentrate a dilute solution of proteins through
>the addition of dry G25 Sephadex. I was wondering if anyone who has used
>this method could comment on how effective it is or if there are any
>disadvantages (like binding of the protein to the beads) that I should be
>aware of. Also, any alternative suggestions for a gentle method of
>concentrating proteins would be appreciated. Thanks.
I've use the Sephadex method when I've had to concentrate proteins already
within 100% Ammonium Sulfate or when glycerol is present. The method
works, but it takes a fair amount of Sephadex to achieve a decent
concentration. I'm not sure of the losses, but they must be there (I'm
usually working with crude mixtures). The biggest hassel is working out an
appliance to do the actual concentrating. I prefer using syringes with
polyethylene frits forced to the bottom and spinning them in 15 or 50 mL
Falcon tubes (plastic top with a hole cut for the syringe which "floats"
above the angled bottom).
A somewhat slow, but reliable method, is to dialyze against polyetheylene
glycol solutions (7 to 50%). The method is a bit slow, requires dialysis
tubing of 12,000 m. wt. or less, but can concentrate into the hundreds of
grams/liter range. Be aware that it's best to simply make the buffer you
want then add the PEG -- making a 20% PEG solution in 1 liter with other
components at nominal concentrations means the dialysis bag will ultimately
contain the buffers/salts at 20% elevated levels.
Eric Larson | University of Illinois at Urbana-Champaign
USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419
Fidonet: 1:233/4.1 | My opinions are my own, but correct :-)
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