Bio-Rad ReadyPrep™ Protein Extraction Kit (Membrane II) - Manual (Page 12)

5. Remove and dilute the supernatant directly into a
beaker or bottle containing 60 ml of the ice-cold
Membrane Protein Concentrating Reagent. Stir the
suspension slowly on ice for 60 min.
6. Transfer the sample to ultracentrifuge tubes and
centrifuge at 100,000 x g for 60 min at 4°C to
pellet the membranes and membrane proteins.
7. Remove the tubes from the rotor and mark the
position of the pellet. Carefully decant and discard the
supernatant.
8. Wash each pellet briefly with 3 ml of cold Lysis Buffer.
Do not disturb the pellet when adding the buffer. Allow
the buffer to sit on ice, covering the pellet, for 1–2 min
before decanting (longer incubation times may be
required for unusually large pellets). Typically, the wash
will easily decant without dislodging the pellet.
However, if any of the pellet dislodges or is seen float-
ing, then the sample should be re-centrifuged at
>20,000 x g for 20–30 min at 4°C.
9. Repeat this wash step using 3 ml of Lysis Buffer per
pellet.
10. Let the tube stand on ice for 1 min and remove any
liquid collected at the bottom of the tube by using a
pipet.
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