Bio-Rad ReadyPrep Sequential Extraction Kit User Manual
Page 10
3
Vortex the mixture for 5 min. For some samples, it may be
necessary to also sonicate the suspension or to aspirate it
through a fine-gauge needle to solubilize the protein.
4
Centrifuge the mixture to give a firm pellet and a clear
supernatant.
5
Recover the supernatant and determine its protein
concentration. The modified Bio-Rad protein assay procedure
shown below is recommended for determining protein
concentrations in extraction solution 2. Store the supernatant
frozen until it is used in 2-D PAGE.
6
Wash the pellet from Step 4 twice with extraction solution 2,
determine the protein concentrations in the washes and
discard them.
7
Dilute the supernatant from Step 5 to standard protein loads
for 2-D PAGE (roughly 1 µg/µl) with extraction solution 2.
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.
9
Discard unused extraction solution 2.
Extraction 3
1
Prepare extraction solution 3 by making a 1:100 dilution of
reducing agent TBP (to 2 mM) into a quantity of Reagent 3.
Mix 10 µl of reducing agent TBP with each 1 ml of Reagent 3
that is used.
8
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