In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Summary

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Abstract

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix.

Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

Protocol

I. Preparation for fractionation

This section describes the preparation of poly-L-Lysine coated microscope coverslips and the attachment of cells prior to fractionation. If required the cells can be transiently transfected with protein expression vectors either before or after attachment.

A. Preparation of poly-L-Lysine coated coverslips

1. Prepare a solution of 1mg/ml poly-L-Lysine in distilled water.
2. Coat clean coverslips with poly-L-Lysine by incubating them in the solution for at least 1 hour on a rocking platform at 22°C.
3. Wash the coated coverslips with sterile distilled wate....

Discussion

Common problems and suggestions:

All or most of the cells are lost during washing. During washing steps liquids must be added slowly to the side of the 6-well plate avoiding the coverslip. Similarly liquids should be removed by carefully tilting the plate and slowly pipetting off excess fluid. Cell adhesion can be increased using poly-L-Lysine coated coverslips.

Genomic DNA is not completely digested. For some cell types the DNAse I digestion step may need to be exte.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSA	Chemical	Sigma-Aldrich	A9647-100G
DNase I	Chemical	Sigma-Aldrich	DN25-1G
poly-L-Lysine	Chemical	Sigma-Aldrich	P-8920
Trypsin	Chemical
EGTA	Chemical	Sigma-Aldrich	E4378-25G
Fomaldehyde	Chemical	VWR	284216N
PIPES	Chemical	Sigma-Aldrich	P-6757
Sucrose	Chemical	Fisher Scientific	S/8600/53
Triton X-100	Chemical	Sigma-Aldrich	T-6876
Mounting Medium with DAPI	Chemical	Vector Laboratories	H-1200
Fugene 6 Transfection Reagent	Chemical	Roche Group	11814443001
Histone H1	Antibodies	Santa Cruz Biotechnology, Inc.	SC-8030
Lamin A/C	Antibodies	Santa Cruz Biotechnology, Inc.	SC-20681
Tubulin-α	Antibodies	Santa Cruz Biotechnology, Inc.	SC-32293