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Published on: February 25, 2011
Two-dimensional electrophoresis reveals a nuclear matrix-associated nucleolin complex of basic isoelectric point
J Gotzmann1, A Eger, M Meissner
1Institute of Tumor Biology-Cancer Research, University of Vienna, Austria.
Electrophoresis
|April 4, 1998
Summary
Researchers identified a novel form of nucleolin (a protein) associated with the nuclear matrix. This nuclear matrix-bound nucleolin interacts with ribonucleoproteins and a basic component, resisting standard dissociation methods.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Nuclear matrix and intermediate filaments serve crucial structural roles in eukaryotic cells.
- Nucleolin is a well-characterized nucleolar protein involved in various cellular processes.
- Understanding protein associations within the nuclear matrix is key to deciphering nuclear organization.
Purpose of the Study:
- To characterize a novel protein recognized by a monoclonal antibody raised against nuclear matrix scaffolds.
- To investigate the properties and localization of this antigen, particularly its relationship with nucleolin.
- To elucidate the composition and interactions of nuclear matrix-bound proteins.
Main Methods:
- Generation of a monoclonal antibody against nuclear matrix/intermediate filament scaffolds.
- Immunoblotting and immunofluorescence microscopy to detect and localize the antigen.
- Partial peptide sequencing for protein identification.
- Two-dimensional gel electrophoresis to analyze protein properties and fractions.
- Fractionation of cellular components and solubilization studies using RNAse and SDS.
Main Results:
- A monoclonal antibody recognized a ~100 kDa protein in human cells and tissues, localized to nucleolar structures.
- Peptide sequencing indicated the antigen is homologous to human nucleolin.
- Two forms of the antigen were detected by 2D electrophoresis: one acidic (pI 5.5-6.1) and one basic (pI 8.5-9).
- The acidic form was soluble, while the basic form was nuclear matrix-bound and RNAse-sensitive.
- RNAse treatment released the basic component, which, upon SDS treatment and re-analysis, shifted to the acidic pI.
Conclusions:
- The nuclear matrix harbors a nucleolin-like protein associated with ribonucleoproteins.
- This nuclear matrix-bound nucleolin exists in a complex with a basic component, resistant to standard dissociation.
- RNAse treatment suggests the involvement of RNA in maintaining the integrity or association of this nucleolin complex within the nuclear matrix.
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