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.

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