FractionPREPTMCell Fractionation System

Introduction:

This FractionPREP cell fractionation system is designed to provide reproducible extraction of four subcellular protein fractions (cytosol, nucleus, membrane/particles, and cytoskeletal fractions) from a single mammalian sample. The method is fast and simple, takes only 2 hours, and no ultracentrifugation involved. The four protein fractions obtained are suitable for many subsequent applications, such as 1-D or 2-D gel, enzyme activity assays, gel shift assay, and Western blotting.

General considerations and reagent preparation:

After opening the kit, you can store buffers at + 4 ° C or –20 ° C. Store protease inhibitor Cocktail and DTT at –20 ° C.
Before starting the procedure, prepare enough Extraction Buffer Mix (EB-Mix) for your Experiment: Add 2 µl of protease inhibitor cocktail and 2 µl of DTT to 1 ml of CEB, SEM-A, and NEB, individually.
Be sure to keep all buffers on the ice at all times during the experiment. All centrifugation is recommended to perform the procedures at 4 ° C.
The following protocol is described for the fractionation of 4 – 8 x 106 cells. If there are more cells used for fractionation, scale volumes proportionally.

FractionPREP fractionation protocol:

Collect cells (4 – 8 x 106) by centrifugation at 700 x g for 5 min. Wash cells with 5-10 ml of ice-cold PBS and centrifuge at 700 x g for 5 min. If using fresh tissue paper, cut it (~ 400 mg) into small pieces, add frozen PBS (1-2ml) and homogenize in a manual tissue homogenizer. Pellet the cells by centrifugation at 500 x g for 5 minutes and remove the supernatant.
Resuspend the cell pellet in 1 ml of ice-cold PBS and transfer the cells to a microcentrifuge tube. Spin for 5 min at 700 x g and remove the supernatant.
Resuspend the pellet in 400 µl of Cytosol Extraction Buffer-Mix (CEB-Mix containing DTT and protease inhibitor cocktail). Pipette several times to mix well with cells. Incubate sample on ice for 20 minutes by tapping 3-4 times every 5 minutes.
Centrifuge the sample at 700 x g for 10 min. Collect the supernatant (this is cytosolic Fraction). Keep on ice.
Resuspend pellet in 400 µl Membrane Extraction Buffer A chilled mix (SEM-A Mix containing DTT and Protease Inhibitor Cocktail). Pipette several times and shake sample for 10-15 seconds to mix well.
Add 22 µl Membrane Extraction Buffer B, vortex for 5 seconds. Incubate on ice for 1 min.
Vortex for 5 seconds again and centrifuge for 5 min at 1000 x g (3400 rpm).
Immediately transfer the supernatant to a clean pre-chilled tube (this is Membrane / Particle Fraction). Keep on ice.
Resuspend the pellet in 200 µl of ice-cold nuclear extraction buffer (NEB-Mix containing DTT and protease inhibitor cocktail), shake for 15 seconds, keep on ice for 40 minutes with constant vortex for 15 seconds every 10 minutes.
Centrifuge the sample at full speed in a microcentrifuge for 10 minutes.
Transfer the supernatant to a clean pre-cooled tube (this is a nuclear fraction). The pellet is the Cytoskeletal Fraction. The cytoskeletal fraction can be dissolved in 100 µl 0.2% SDS containing 10 mM DTT or dissolve directly in SDS-PAGE sample buffer
Store all fractions at –80 ° C for future use.