Proteomics/Protein Separations- Electrophoresis/Types of Gel Electrophoresis/QPNC-PAGE
Quantitative Preparative Native Continuous PolyAcrylamide GelElectrophoresis is a technique to isolate active or native metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides) in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures.
The gel is polymerized for a time period of 69 hr at room temperature to fully-polymerize it. That results in a gel which is homogeneous, mechanically stable and free of monomers or radicals. Therefore, the interactions of the gel with the biomolecules can be neglected. The separated metalloproteins are not dissociated into apoproteins and metal cofactors. Highly purified metalloproteins can be isolated in quantitative amounts from different PAGE fractions. The active structures or conformation of the isolated proteins do not change by using QPNC-PAGE.
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. Although the pH value (pH 10.00) of the electrophoresis buffer does not correspond to a physiological pH within a cell or tissue, the proteins are eluted continuously by a physiological buffer solution (pH 8.00) and isolated in different fractions. The separation system including the electrophoresis chamber and a fraction collector is cooled in a refrigerator at 4°C.
Identification and Quantitation
Cd, Zn, Cu, Ni, Pd, Co, Fe, Mn, Pt, Cr, Mo and other metal cofactors can be identified and quantified by ICP-MS. Because of high purity and optimized concentration of the separated metalloproteins in specific PAGE fractions, the related structures of these analytes can be elucidated by using solution NMR spectroscopy under non-denaturing conditions.
QPNC-PAGE combined with biological mass and NMR spectrometries provide valuable information about the metabolisms of important metal cofactors in biological systems and the proper folding of metallochaperones in conformational diseases (e.g., Alzheimer's or related diseases). The quantitative analysis of metallochaperone proteins in biofluids (e.g., blood, cerebrospinal fluid) helps in the clinical investigations concerning the structure-function relationships of biologically-active metal cofactor-containing chaperones in those protein-misfolding diseases.
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- Kastenholz, B. 2006. Comparison of the electrochemical behavior of the high molecular mass cadmium proteins in Arabidopsis thaliana and in vegetable plants on using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE). Electroanalysis 18, 103-106.
- Kastenholz, B. 2004. Preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE): An efficient method for isolating cadmium cofactors in biological systems. Anal. Lett. 37, 657-665.