Proteomics/Protein Separations- Electrophoresis/QPNC-PAGE
Quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is an electrophoresis technique to isolate native or active metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures. Some examples of metalloproteins are metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, and metallopeptides.
Gel and Buffer Characteristics
The gel is polymerized for a time period of sixty-nine hours at room temperature. That results in a gel which is mechanically stable, homogeneous, and free of monomers or radicals, and therefore, only negligible interactions of the gel with the biomolecules occurs. The metalloproteins are not dissociated into metal cofactors and apoproteins during electrophoresis, and purified metalloproteins are isolated in quantitative amounts from different PAGE fractions. Due to the specific characterstics of the gel, the conformation or active structures of the isolated proteins are not changed by this method.
The chemical stability of a specific metalloprotein can be investigated and confirmed by using molecular exclusion chromatography.
The buffer used for electrophoresis contains 20 mM Tris-HCl, 1 mM NaN3 and has a pH 10. Due to the specific properties of the gel and buffer solution, most proteins in the sample are charged negatively and migrate from the cathode to the anode in the electric field. The proteins are eluted continuously by a physiological buffer solution and isolated in different fractions.
Analysis and Applications
Metal cofactors eluted in different PAGE fractions can be identified and quantified by mass spectrometry. Since the pure metalloproteins are separated under non-denaturing conditions, the structures of these proteins can be obtained by solution NMR spectroscopy.
Improper folding of metalloproteins results in degenerative conditions like Alzheimer's disease. The quantitative analysis of metallochaperone proteins in biofluids helps in the clinical investigations concerning the structure-function relationships of biologically-active metal cofactor-containing chaperones in those protein-misfolding diseases. QPNC-PAGE combined with biological mass and NMR spectrometries provides valuable information about the metabolisms of important metal cofactors in biological systems.
^ "QPNC-PAGE: The Standard Protocol." http://www.grin.com/en/e-book/202981/qpnc-page-the-standard-protocol ^ Kastenholz, B. "New Hope for the Diagnosis and Therapy of Alzheimer's Disease." Protein and Peptide Letters(2007) 14:389-393.