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Updated: Aug 11, 2026
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Published on: February 24, 2011
Screening for beta-poly(L-malate) binding proteins by affinity chromatography
Thomas Göttler1, Eggehard Holler
1Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, D-93040 Regensburg, Germany.
Abstract:
Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes.
Insights
Poly(beta-L-malic acid), a slime mold polymer, binds to proteins involved in cell mobility. Researchers identified these proteins using affinity chromatography, revealing a large complex protein in Physarum polycephalum.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Poly(beta-L-malic acid) is a unique polymer found in myxomycetes (slime molds).
- It plays a role in organizing protein mobility within the giant multinucleated plasmodia of these organisms.
Purpose of the Study:
- To identify proteins that bind to poly(beta-L-malic acid) in Physarum polycephalum.
- To characterize the properties and cellular localization of these poly(beta-L-malic acid)-binding proteins.
Main Methods:
- Affinity chromatography using 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid).
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing PAGE for molecular mass and complex analysis.
- Western blotting and silver staining for protein detection.
- 2-dimensional non-denaturing PAGE/SDS-PAGE for complex/subunit analysis.
- Gel shift assays to assess DNA binding.
Main Results:
- Identification of poly(malate)-binding proteins in cellular extracts of Physarum polycephalum.
- A significant 370 kDa protein complex was found, dissociating into 11 subunits (11-29 kDa).
- Poly(malate)-binding proteins were most abundant in nuclei, followed by cytoplasm and membranes.
- Proteins demonstrated binding to nucleic acids in gel shift experiments.
- Poly(malate) is proposed as a functional equivalent to eukaryotic aspartate repeats.
Conclusions:
- Poly(beta-L-malic acid) interacts with specific proteins, particularly within the nucleus.
- These proteins are complex and exhibit nucleic acid binding properties.
- Poly(malic acid) may serve a structural or functional role analogous to aspartate repeats in eukaryotes.
