Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 11)

2 Use extraction solution 3 to solubilize proteins in the pellet
from Extraction 2. Use approximately the same volume of
extraction solution 3 as was used of extraction solution 2.
Empirically determine the best volume for the third extraction.
3 Vortex and centrifuge as described above.
4 Recover the supernatant and determine its protein content.
The modified Bio-Rad protein assay procedure shown below
is recommended for determining the protein content in
extraction solution 3. Store the supernatant frozen until it is
used in 2-D PAGE. It may be informative to wash the pellet
and determine the protein concentration in the wash solution.
5 The pellet can be extracted with SDS to yield highly insoluble
proteins as described in [1].
6 Dilute the supernatant from Step 4 to standard protein loads
for 2-D PAGE (roughly 1 µg/µl) with extraction solution 3.
7 Because of the ionic nature of the extracts, it is beneficial to
begin the isoelectric focusing run at low voltage. For example,
limit the voltage to 250 V for 1 hr then 500 V for 1 hr before
beginning a ramp to the final focusing voltage. Use paper
wicks under the electrodes to capture impurities in the
samples.
8 Discard unused extraction solution 3.
The three extracts can have different conductivity. With some
isoelectric focusing chambers, best reproducibility is achieved
by focusing the three extracts separately.
9
4006167b.qxd 08/28/2000 6:43 PM Page 9