Bio-Rad ReadyPrep™ Protein Extraction Kit (Membrane I) User Manual

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7. Add 0.5 ml of prechilled buffer M2 (the same volume

as used in step 3) to the tube containing the bottom
phase. Vortex and incubate as described in step 3.
Repeat steps 4 through 6. Again remove the top layer
and pool with the top layer that was collected earlier.
Label this tube of pooled top layers Hydrophilic Protein
Fraction (Refer to the note in step 9 for further
instructions on storing this protein fraction).

8. If there is any debris at the interface, either remove it

and set aside for later analysis or discard.

9. Collect the bottom phase and transfer to a new tube.

Label this tube Hydrophobic Membrane Protein
Fraction (Refer to the note below for further
instructions on storing this protein fraction). The
sediment, which is an additional source of membrane
proteins, can be saved and stored at -70°C for later
analysis (step 11).

Note: If no further downstream applications are to be
immediately performed, aliquot and quick-freeze the
hydrophilic and hydrophobic membrane protein fractions on
dryice and then store at -70°C. Also, to avoid repeated
freeze-thawing of the sample, it is often convenient at this
stage to set aside a 5–10 µl aliquot of each protein fraction
before freezing for protein quantitation. This amount is
sufficient for the RC DC protein assay (catalog #500-0121
and #500-0122).

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