Bio-Rad ReadyPrep™ 2-D Cleanup Kit - Manual (Page 9)

bottom of the tube. Use a pipet to carefully remove the
remaining supernatant.
7. Add 40 µl of wash reagent 1 on top of the pellet.
Position the tube in the centrifuge as before and
centrifuge at maximum speed (> 12,000 x g) for 5 min.
Note: A precipitate may form along the tube wall. In these
cases, vortex and/or pipet wash solution over the pellet
several times to ensure entire pellet is thoroughly washed.
8. With a pipet, remove and discard the wash.
9. Add 25 µl of ReadyPrep proteomic grade water or
other ultrapure water on top of the pellet. Vortex the
tube 10–20 sec. Protein pellets may disperse, but will
not dissolve in the water.
10. Add 1 ml of wash reagent 2 (prechilled at -20°C for at
least 1 hr) and 5 µl of wash 2 additive. Vortex the
tube for 1 min.
Notes: Protein pellets will not dissolve in wash reagent 2.
If wash reagent 2 is not completely chilled, quantitative
recovery may be affected.
11. Incubate the tube at -20°C for 30 min. Vortex the tube
for 30 sec every 10 min during the incubation period.
12. After the incubation period, centrifuge the tube at top
speed for 5 min to form a tight pellet. Remove and
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