ammonium sulfate precipitation of proteins

[larson eric]

bipin dalmia <dalmiabk at phibred.com> writes:

>the protein that i am precipitating does precipitate at 70% ammonium
>sulfate saturation from a crude extract. the 3 mg/ml solution was a
>pooled fraction from an ion-exchange step that was greater than 80% pure.
===========
 
It's possible your protein in the crude extract either needed other
proteins for good ppt. or the presence of membraneous components for good
sedimentation (or many other possibilities best discussed over coffee :-).
 
How to recover what you have?  If it were me, I'd put the cloudy ppt. in a
dialysis bag (maximum of 10,000 to 13,000 kDa), then place in a large
volume of solution without ammonium sulfate but with my buffer of choice.
 
Once dialysis had resolubilized the protein (usually does), I'd put
polyethylene glycol 10,000 (newly purchased Sigma is clearly best versus 
old stocks on the shelf) to 15 to 20% by volume (i.e. 200 g PEG 10K for 1
liter to make 20%).  The PEG should concentrate your protein within the
dialysis bag.  If you do the PEG dialysis procedure too long, the protein
can reach saturation (i.e. ppt. out of solution).  If this occurs, change
the buffer to one without PEG and continue dialysis until the bag picks up
enough water to solubilize your protein.  For what it's worth, I've used
PEG concentration to make several hundred milligram/mL solutions of
protein.

-- 
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 :-)