Bio-Rad ReadyPrep™ 2-D Cleanup Kit - Manual (Page 8)

1. Transfer 1–500 µg of protein in a final volume of
100 µl into a 1.5 ml microcentrifuge tube.
Note: Sample quantities > 500 µg of protein may reduce the
efficiency of the cleanup leading to poor-quality IEF.
2. Add 300 µl precipitating agent 1 to the protein
sample and mix well by vortexing. Incubate on ice for
15 min.
Note: When adding solution, do not touch protein sample
with the pipet tip. The protein may precipitate on the tip
causing sample loss.
3. Add 300 µl precipitating agent 2 to the mixture of
protein and precipitating agent 1. Mix well by
vortexing.
Note: When adding solution, do not touch protein sample
with the pipet tip. The protein may precipitate on the tip
causing sample loss.
4. Centrifuge the tube(s) at maximum speed
(> 12,000 x g) for 5 min to form a tight pellet. Remove
the tube promptly once the centrifuge stops so that
the pellet does not disperse.
5. Without disturbing the pellet, remove and discard the
supernatant using a pipet.
6. Position the tube in the centrifuge as before (i.e., cap
hinge and protein pellet facing outward) and centrifuge
for 15–30 sec to collect any residual liquid at the
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