QPNC -PAGE ( short for: . Quantitative preparative native continuous polyacrylamide gel electrophoresis ) is a standardized variant of the native gel electrophoresis. This analytical method of biochemistry and bioinorganic chemistry is used for the separation of charged molecules in a homogeneous electric field and allows the quantitative separation and isolation of metalloproteins from human, plant or animal samples with high resolution. Proteins are separated according to the isoelectric points and analyzed by NMR spectroscopy. The method thus makes a contribution to the structure determination of native and denatured metalloproteins and isoforms and their interactions in complex protein mixtures.
The separation of the proteins is carried out in a commercially available electrophoresis for preparative polyacrylamide gel electrophoresis ( PAGE). It is made up of various components, the upper and lower electrophoresis and the gel column with elution and cold finger. In addition, the electrophoresis device, a DC voltage source, a peristaltic pump for transport of the eluent to a fraction collector, a UV detector, a recorder and a pump for the cooling circuit of the bottom of the gel electrophoresis are used for cooling. All devices except voltage source and recorder are cooled during electrophoresis in a refrigerator constant at 4 ° C ( see figure).
Electrophoresis and gel
When electrophoresis is continuous at QPNC -PAGE a special buffer solution, a mixture of 20 mM Tris- HCl and 1 mM NaN 3 as a biocide, selected with the pH 10.00. Continuously in this context that both the anode buffer and the cathode buffer and the buffer for the preparation of the polyacrylamide gel of the same chemical composition and concentration. Most of the proteins of a living organism are negatively charged due to their isoelectric point at the pH value of 10.00, and therefore migrate in the electric field from the cathode to the anode. The flow of current between the cathode and anode causes the buffer solution, the electrophoretic movement of charged molecules, and as a side reaction and electrolysis operations, with the associated electrode reactions. This produces at the cathode hydrogen gas and oxygen at the anode is formed mainly in a 2:1 ratio, to a lesser extent, elemental chlorine. At the anode, at the same time hydrogen ions are generated in these electrochemical processes, which react with Tris Tris molecules monovalent ions. The positively charged Tris- ions migrate through the gel towards the cathode, where they react with hydroxide ions to Tris molecules and water. The Tris -based buffering causes a constant pH value in the buffer system. The resulting gel in the electrophoresis in the heat is removed by cooling on the one hand externally, and on the other hand by an internal cooling circuit in the Electrophoresis.
The polyacrylamide gel must be made on the basis of the technical specifications of electrophoresis used before each run fresh. The preparation is carried out in principle as in the polyacrylamide gel electrophoresis, but in a QPNC - PAGE resolving gel with no stacking is used. The final gel has in comparison to other gel electrophoresis over a relatively large pore structure having a total monomer content of 4 % T (T = total amount of acrylamide and N, N -methylene bisacrylamide in the gel [w / v] ) with a cross-linking of 2.67 % C (C = N ratio, N-Methylenbisacrylamid/Acrylamid [w / w] ). It has a height of 40 mm with an inner diameter of 28 mm. As the catalyst for the polymerization of 0.5 ul TEMED / ml of gel are used, the proportion of APS based on the total monomer as a radical initiator of the gel is 1.25 % [w / w].
The gel outside of the electrophoresis on a special device where the gel column and the cold finger are secured molded. It requires a total of 69 hours to complete the polymerization at room temperature, it is an exothermic reaction. The heat generated is dissipated by means of the internal cooling circuit of the electrophoresis. This procedure causes no free monomers are more in the gel, whereby the reactions of gel components ( gel matrix ) can be eliminated with the analyte to be isolated ( proteins). Molekularsiebeffekte or other interactions with the gel matrix, the analyte (see gel electrophoresis) can be approximately considered because the pore size and uniformity of the gel as compared with a separation gel with a different native gel electrophoresis or SDS-PAGE to be negligible. The gel is mechanically stable and therefore manageable. The polymerization of the gel associated with the compression of the gel pores is crucial for the reproducibility of the elution times of metalloproteins in the electropherogram.
Samples to be analyzed are mixed prior to electrophoresis with 10% [ v / v] glycerol in order to avoid denaturation of the ingredients in the sample application. The sample itself, the total protein content should not be more than 0.5 mg, thus receives a higher density and can be controlled under the cathode buffer to apply to the gel surface.
The isolation of metalloproteins from complex biotic mixtures ( eg, blood, cerebrospinal fluid ) is particularly effective with this method because the fraction of uncharged polar molecules (eg sugar) and the positively charged molecular ions (cations) does not migrate through the gel, but dissolved in the cathode buffer remains. Which are present in the sample negatively charged molecular ions (anions ) can migrate through the gel towards the anode and are eluted continuously in the elution. In this process, first the small inorganic and organic anions are eluted and then collected the proteins in fractions. Consequently, the analytes to be removed from the gel.
The properties of the QPNC -PAGE also cause an electrophoretic run on either the size ( molecular mass ) or forms ( form ) to be isolated proteins affect a separation. Instead the insulation is carried out of the separated proteins ( metalloproteins, proteins with organic cofactors and without cofactors ) and peptides only in accordance with the isoelectric points (pI). Native and denatured molecules of a protein complex have different isoelectric points, the rate of migration of a protein through the gel directly from the respective pI depends. Isomers of proteins as well as native and denatured metalloproteins, for example, are present together in a sample, are therefore presented separately in an electropherogram.
The separated respective molecules move with a constant speed different than annular protein bands by the gel and then a physiological buffer (20 mM Tris-HCl, 1 mM NaN3, pH 8.00) are reproducibly eluted and isolated fractions. In the case of a separation, neither the present metal - protein complexes dissociated, altered the native conformations. The resolving power of QPNC -PAGE for metal cofactors ( eg, Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr or Cd) moves in the physiologically effective range of about 1 ng / ml. After an identification and absolute quantification of metal cofactors with the ICP -MS ( short for: . Inductively coupled plasma mass spectrometry ), an isolated metal protein are fed quantitatively due to its high purity and optimized concentration of NMR spectroscopy.
With the help of NMR spectroscopy as multidimensional protein structures solved by native and misfolded proteins and with the help of bioinformatics structure-function relationships for various metalloproteins ( in certain fractions eg Metallochaperone, prions, metalloenzymes, metallopeptides (eg beta -amyloid u a.) ) are determined. Proteins and their interactions can thereby be precisely analyzed. Particularly important is this approach for clinical diagnosis and therapy in the field of protein misfolding diseases whose causes lie in a misfolding of proteins, including, for example, Alzheimer's disease or Creutzfeldt -Jakob disease count. Of particular importance for the development of new drugs for certain protein misfolding diseases have now achieved medicinal plants and crops. On an industrial scale for this purpose pharmacologically active recombinant proteins ( see Molecular pharming ) or other biologically active plant substances generated in the body whose action (English: "relative biochemical impact" ) detected by means of a strategy consisting of analytical, biochemical and biophysical methods can be. The interdisciplinary nature of the methodology used to reduce the chance that there are artifacts in the corresponding results.