Quick and easy soluble cytosol vs insoluble cytosol sample prep

Overview

This is a quick and easy method to prepare soluble cytosolic extracts and insoluble extracts. It is often used to study polymerisation of actin monomer (G-actin, soluble) into actin polymer and stress fibres (F-actin, insoluble). It can also be used to examine cytosol-to-nucleus translocation of proteins such as transcriptional co-factors, which are often soluble in the cytosol but appear in the insoluble fraction when nuclear.

For a classical nuclear vs cytoplasmic split by hypotonic lysis and Dounce homogenisation, see cell fractionation: nuclei extraction.

Procedure

1. Seed cells into appropriate culture plates (e.g. 6-well plates).
2. Stimulate or treat with reagents as required.
3. Remove media.
4. Add 300–400 µl of cytosol lysis buffer and rock the plate gently for 5 min in a cold room.
5. Collect lysate (Triton-soluble / cytosol fraction).
6. Add SDS lysis buffer (same volume as used for cytosol) to the well. Rock at room temperature so lysis buffer covers the entire bottom of the well.
7. Collect SDS lysate (nuclear / Triton-insoluble fraction).
8. For western blot analysis, add an equal volume of 2× SDS buffer to the soluble Triton extract.
9. Add bromophenol blue to samples to give a medium blue colour for easy gel loading and monitoring of electrophoresis.
10. Add β-mercaptoethanol to the samples at 2.5 µl/ml.
11. Heat samples to 95 °C for 5 minutes.
12. Load double the volume of the soluble Triton extract sample to maintain an equal 1:1 loading of Triton-soluble to SDS extracts on the gel.

Cytosol lysis buffer (10 ml)

Triton-insoluble / nuclear SDS lysis buffer

• 50 mM Tris pH 6.8
• 2% SDS
• 20% glycerol

Use the same volume per well as cytosol lysis buffer. For western blotting, combine fractions with 2× SDS, β-mercaptoethanol, and bromophenol blue as in the procedure above.