Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 9)

with the Bio-Rad protein assay (Bradford), catalog # 500-0001,
or the modified assay shown below.
5 Wash the insoluble pellet from Step 3 twice with the same
volume of Reagent 1 used in Step 1. It is valuable to
determine the protein concentrations in the washes. Discard
the washes.
6 Store the supernatant frozen until it is used in 2-D PAGE.
7 Dilute the supernatant from Step 6 to standard protein loads
for 2-D PAGE (roughly 1 µg/µl) with extraction solution 2
[Reagent 2 containing a 1:100 dilution (2 mM) of reducing
agent TBP].
8 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 up to the final focusing voltage. Use paper
wicks under the electrodes to capture impurities in the
samples.
Extraction 2
1 Prepare extraction solution 2 by making a 1:100 dilution of
reducing agent TBP (to 2 mM) into a quantity of Reagent 2.
Mix 10 µl of reducing agent TBP with each 1 ml of Reagent 2
that is used.
2 Use extraction solution 2 to solubilize proteins in the pellet
from Extraction 1 (Extraction 1, Step 5). Use a volume of
extraction solution 2 that is about half the volume of Reagent 1
used in the first extraction. The best volume to use should be
determined empirically.
7
4006167b.qxd 08/28/2000 6:43 PM Page 7