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Bio-Rad ReadyPrep Sequential Extraction Kit User Manual

Page 10

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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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