Premium
Two‐dimensional electrophoresis reveals a nuclear matrix‐associated nucleolin complex of basic isoelectric point
Author(s) -
Gotzmann Josef,
Eger Andreas,
Meissner Michael,
Grimm Rudolf,
Gerner Christopher,
Sauermann Georg,
Foisner Roland
Publication year - 1997
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.1150181421
Subject(s) - nucleolin , nuclear matrix , isoelectric point , immunoprecipitation , rnase p , ribonucleoprotein , microbiology and biotechnology , isoelectric focusing , antigen , nuclear protein , chemistry , sodium dodecyl sulfate , cell nucleus , gel electrophoresis , biology , biochemistry , cytoplasm , chromatin , rna , nucleolus , enzyme , dna , gene , genetics , transcription factor
A monoclonal antibody was raised against a salt‐extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an ∼ 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two‐dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (p I ) 5.5–6.1, characteristic for nucleolin, and at p I 8.5–9. Whereas the protein focusing at acidic p I was found in Triton X‐100‐soluble cellular fractions, the antigen focusing at basic p I was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic p I in two‐dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse‐released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix‐derived antigen was positioned at p I 5–6. The present data indicate that the nuclear matrix‐bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.