2D DIGE Analysis of Serum After Fractionation by ProteoMiner Beads - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 187

Fig. 1. Protein profi les of crude and fractionated serum on a 13% SDS-PAGE gel. Lane M
is molecular weight marker. Lanes E1-E4 are fractions derived from sequential elution
of bound proteins from the ProteoMiner™ column using 8 M urea, 2% (w/v) CHAPS, and
5% acetic acid. Lane EC is a combined pool of E1-E4. Lane CS is loaded with 1 m L of
unfractionated serum, while lanes E1-E3 are loaded with 0.2 m L, and lane E4 is loaded
with 0.6 m L of the sequential eluates.

3.3. Sample
Precipitation Using
2D Clean-Up Kit

All steps should be carried out on ice (4-5°C) at all times unless
otherwise stated according to the manufacturer's manual.

1. Prechill the wash buffer prior to use for at least 1 h at −20°C.
2. Transfer the ProteoMiner™ fractionated sample into a 1.5-mL
microcentrifuge tube (see Note 5). Ensure that the tube capac-
ity is at least 8 times greater than the sample volume.
3. For each sample, add 3 volumes of precipitant to 1 volume of
sample. Vortex and incubate on ice for 15 min.
4. Then, add 3 volumes of coprecipitant to the mixture and
vortex briefl y to mix.
5. Centrifuge at a maximum centrifugal force of at least 12,000 × g
for 5 min.
6. Remove the tube immediately and discard as much of the super-
natant as possible using a pipette. Avoid disturbing the pellet.
7. Next, carefully reposition the tube in the centrifuge as before
and pulse-spin it to bring down the remaining liquid to the
bottom of the tube. Remove all the liquid from the tube.