Ammonium Sulfate Precipitation of Proteins Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Ammonium sulfate precipitation of proteins protocol

The following protocol is for secreted proteins. Modify accordingly for other soluble proteins

1. Spin down cells
2. Pour out supernatant into a flask or beaker
3. Take out a fraction to analyze on gel
4. Add desired amount of ammonium sulfate very slowly (either in a saturated liquid form, or in powder form)
5. Stir for 1 hour
6. Spin down the precipitated protein at 10,000 x g., 30 minutes
7. Resupend pellet in PBS for dialysis (concentrate approximately 1/6x to 1/10x based on the amount of the original media)
8. Take some of the supernatant from the spun down protein for dialysis

Desired
% Saturation     Amount of saturated ammonium sulfate to add (for 10mL media)
10                       1.11ml
20                       2.5ml
30                       4.29ml
40                       6.67ml
50                       10ml
55                       12.22ml
60                       15ml
65                       18.57ml
70                       23.33ml
75                       30ml
80                       40ml
85                       56.66ml
90                       90ml
95                       190ml