Premium
Gel electrophoretic analysis of nuclear matrix fractions isolated from different human cell lines
Author(s) -
Kallajoki Markku,
Osborn Mary
Publication year - 1994
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150150170
Subject(s) - electrophoresis , nuclear matrix , chemistry , chromatography , matrix (chemical analysis) , biochemistry , dna , chromatin
Abstract The nuclear matrix is operationally defined as the structure that remains after nuclei are extracted with nonionic detergent and with high salt and are digested with nucleases. Thus the nuclear matrix protein composition is critically dependent on the isolation conditions. We have compared nuclear matrices isolated from human cell lines by two different methods. First, isolated nuclei were extracted as above to obtain a matrix fraction. This method showed a substantial contamination by cytoplasmic intermediate filaments but immunization of mice resulted in antibodies recognizing nuclei and the mitotic spindle apparatus. Second, a nuclear matrix fraction was made by extracting whole cells as above and dissolving the residue in urea and dialysing against an assembly buffer to precipitate intermediate filament proteins (Fey, E. G. and Penman, S., Proc. Natl. Acad. Sci. USA 1988, 85 , 121–125). Such fractions showed complex protein patterns in silver‐stained two‐dimensional gels for four cell lines: HeLa, MCF‐7, SW13 and the U333CG/343MG glioma line. While some proteins in the nuclear matrix fraction were common to all cell lines, others appeared cell‐line specific. Two‐dimensional gels and the immunoresponse in mice again showed contamination of these preparations with cytoplasmic proteins. These results clearly show the difficulties associated with protein chemical analysis of nuclear matrices: the preparations have substantial cytoplasmic contamination, the polypeptide composition is extremely complex and the yield of individual polypeptides is low. Thus, without further experiments one cannot say which proteins are true nuclear matrix components. In addition, our results underline the value of dissecting the nuclear matrix by analyzing its individual components with monoclonal antibodie, and we show examples of the nuclear staining patterns of three different classes of monoclonal antibodies isolated by this approach.