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Cold Spring Harb Protoc. 2016 Jan 4;2016(1):pdb.prot083758. doi: 10.1101/pdb.prot083758.

Preparation of the Nuclear Matrix for Parallel Microscopy and Biochemical Analyses.

Author information

1
Department of Biology, University of York, Wentworth Way, York YO10 5DD, United Kingdom.

Abstract

Immobilized proteins within the nucleus are usually identified by treating cells with detergent. The detergent-resistant fraction is often assumed to be chromatin and is described as such in many studies. However, this fraction consists of both chromatin-bound and nuclear-matrix-bound proteins. To investigate nuclear-matrix-bound proteins alone, further separation of these fractions is required; the DNA must be removed so that the remaining proteins can be compared with those from untreated cells. This protocol uses a nonionic detergent (Triton X-100) to remove membranes and soluble proteins from cells under physiologically relevant salt concentrations, followed by extraction with 0.5 m NaCl, digestion with DNase I, and removal of fragmented DNA. It uses a specialized buffer (cytoskeletal buffer) to stabilize the cytoskeleton and nuclear matrix in relatively gentle conditions. Nuclear matrix proteins can then be assessed by either immunofluorescence (IF) and immunoblotting (IB). IB has the advantage of resolving different forms of a protein of interest, and the soluble fractions can be analyzed. The major advantage of IF analysis is that individual cells (rather than homogenized populations) can be monitored, and the spatial arrangement of proteins bound to residual nuclear structures can be revealed.

PMID:
26729903
DOI:
10.1101/pdb.prot083758
[Indexed for MEDLINE]

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