QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a universal high-resolution technique applied in biochemistry and bioinorganic chemistry to separate proteins by isoelectric point irrespective of their mass, size and shape. This standardised variant of native gel electrophoresis is used by biologists to isolate active or native metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins or protein isoforms in complex protein mixtures.
Electrophoresis buffer and device
The QPNC-PAGE procedure is accomplished in a commercially available electrophoresis chamber for separating charged biomolecules. Due to the specific properties of the prepared gel and electrophoresis buffer solution (which is basic and contains Tris-HCl and NaN3), most proteins of a biological system are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field.
Although the pH value (10.00) of the electrophoresis buffer does not correspond to a physiological pH value within a cell or tissue type, the separated ring-shaped protein bands are eluted continuously into a physiological buffer solution (pH 8.00) and isolated in different fractions. Most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C. The separation system, including the electrophoresis chamber and a fraction collector, is cooled in a refrigerator. 
Gel and gel properties
The time of polymerization of the gel may directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression of the gels and their pores with the longer incubation times (see Figure Electropherogram). In order to obtain a fully polymerized gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature. As a result, the prepared fresh gel is homogeneous, mechanically stable and free of monomers or radicals. The 4% T, 2.67% C gel is essentially non-sieving and optimal for electrophoresis of proteins that are smaller and larger than 200 ku. Proteins migrate in it more or less on the basis of their free mobility. For these reasons interactions of the gel with the biomolecules can be neglected and the proteins separate cleanly and predictably. The separated metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides) are not dissociated into apoproteins and metal cofactors.
Reproducibility and recovery
The bioactive structures (native or 3D conformation) of the isolated protein molecules do not undergo any significant conformational changes. Active metal cofactor-containing proteins can be isolated reproducibly in the same fraction after a PAGE run (see Figure Electropherogram). A shifting peak in the respective electropherogram may indicate that a denatured metalloprotein is available in the original protein mixture to be separated. Metalloproteins with different molecular mass ranges and isoelectric points have been recovered in biologically active form at a yield of more than 95%.
Quantification and identification
Low concentrations (ppb-range) of Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal cofactors can be identified and absolutely quantified by inductively coupled plasma mass spectrometry (ICP-MS), for example. Because of high purity and optimized concentration of the separated metalloproteins, for example, therapeutic recombinant plant-made pharmaceuticals such as copper chaperone for superoxide dismutase (CCS) from medicinal plants, in specific PAGE fractions, the related structures of these analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions.
Improperly folded metal proteins, for example, CCS or superoxide dismutase (SOD1) present in brain, blood or other clinical samples, are indicative of neurodegenerative diseases like Alzheimer's disease or Amyotrophic Lateral Sclerosis. Active CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ions (e.g., Cu1+/2+, Zn2+, Fe2+/3+) in organisms and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm.
In 2001 Bernd Kastenholz (*1960) discovered a new basic principle of gel electrophoresis at the Forschungszentrum Jülich. This indicates that the time of polymerization of a polyacrylamide gel directly affects the result of a protein purification because the separation properties of a gel are significantly influenced by this parameter. As a consequence, the peak-elution times implying the reproducibility of the separated proteins may vary considerably. Said principle ("69 hr rule") as an integral part of a new technique was applied for patent in 2003, accepted in 2013 (German Patent DE 103 36 540.0) and realized by QPNC-PAGE. Several articles, press releases and conference papers concerning QPNC-PAGE were published from 2006 to 2010. Most of these publications were edited or coauthored by the well-known American scientist and internationally recognized expert in the field of protein electrophoresis David E. Garfin. In the 1970s and the early 1980s Garfin became one of the main investigators in the field of prion research in the team of Stanley B. Prusiner.
- Seelert H, Krause F (2008). "Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels". Electrophoresis 29 (12): 2617–36. doi:10.1002/elps.200800061. PMID 18494038.
- Youn HD, Kim EJ, Roe JH, Hah YC, Kang SO (1996). "A novel nickel-containing superoxide dismutase from Streptomyces spp". Biochemical Journal 318 (Pt 3): 889–96. PMC 1217701. PMID 8836134.
- Fitri N, Kastenholz B, Buchari B, Amran MB, Warganegara FM (2008). "Molybdenum speciation in raw phloem sap of castor bean". Analytical Letters 41 (10): 1773–84. doi:10.1080/00032710802162442. ISSN 0003-2719.
- McLellan T (1982). "Electrophoresis buffers for polyacrylamide gels at various pH". Analytical Biochemistry 126 (1): 94–99. doi:10.1016/0003-2697(82)90113-0. ISSN 0003-2697. PMID 7181120.
- Garfin DE (2009) . "Chapter 29 one-dimensional gel electrophoresis". Methods in Enzymology 463: 497–513. doi:10.1016/S0076-6879(09)63029-9. ISBN 978-0-12-374536-1. PMID 19892189.
- Kastenholz B (2006). "Important contributions of a new quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) procedure for elucidating metal cofactor metabolisms in protein-misfolding diseases – a theory". Protein and Peptide Letters 13 (5): 503–8. doi:10.2174/092986606776819637. PMID 16800806.
- Kastenholz B, Garfin DE (2009). "Medicinal plants: a natural chaperones source for treating neurological disorders". Protein and Peptide Letters 16 (2): 116–120. doi:10.2174/092986609787316234. PMID 19200033.
- Robinson NJ, Winge DR (2010). "Copper metallochaperones". Annual Review of Biochemistry 79: 537–562. doi:10.1146/annurev-biochem-030409-143539. PMID 20205585.
- Garfin DE (2009). "25th Annual Meeting of the American Electrophoresis Society". Expert Review of Proteomics 6 (3): 239–241. doi:10.1586/epr.09.18. PMID 19489696.
- Prusiner SB, Garfin DE, Cochran SP, McKinley MP, Groth DF, Hadlow WJ, Race RE, Eklund CM (1980). "Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent". Journal of Neurochemistry 35 (3): 574–582. doi:10.1111/j.1471-4159.1980.tb03693.x. PMID 6778963.
- Kastenholz B, Garfin DE. "Isolation of Acidic, Basic and Neutral Metalloproteins by QPNC-PAGE". Nature Precedings
- Garfin DE. "Gel Electrophoresis of Proteins". AES Application Foci
- Garfin DE. "Reminiscences About Electrophoresis"
- Garfin DE. "Person Profile".