QPNC-PAGE
QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a bioanalytical, high-resolution and highly accurate technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point. This standardized variant of native gel electrophoresis is used by biologists to isolate biomacromolecules in solution, for example, active or native metalloproteins in biological samples or properly and improperly folded metal cofactor-containing proteins or protein isoforms in complex protein mixtures.
Introduction
Proteins perform several functions in living organisms, including catalytic reactions and transport of molecules or ions within the cells, the organs or the whole body. The understanding of the processes in human organisms, which are mainly driven by biochemical reactions and protein-protein interactions, depends to a great extent on our ability to isolate active proteins in biological samples for more detailed examination of chemical structure and physiological function. This essential information can imply an important indication of a patient's state of health.As about 30-40% of all known proteins contain one or more metal ion cofactors, especially native metalloproteins have to be isolated, identified and quantified after liquid biopsy. Many of these cofactors play a key role in vital enzymatic catalytic processes or stabilize globular protein molecules. Therefore, the high-precision electrophoresis and other native separation techniques are highly relevant as initial step of protein and trace metal speciation analysis, subsequently, followed by mass spectrometric and magnetic resonance methods for quantifying and identifying the soluble proteins of interest.
Method
Separation and buffering mechanisms
In gel electrophoresis proteins are normally separated by charge, size, or shape. The aim of isoelectric focusing, for example, is to separate proteins according to their isoelectric point, thus, according to their charge at different pH values. Here, the same mechanism is accomplished in a commercially available electrophoresis chamber for separating charged biomolecules, for example, superoxide dismutase or allergens, at continuous pH conditions and different velocities of migration depending on different isoelectric points. The separated proteins elute sequentially, starting with the lowest and ending with the highest pI of the present protein molecules.Due to the specific properties of the prepared gel and electrophoresis buffer solution, 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. At the anode, electrochemically-generated hydrogen ions react with Tris molecules to form monovalent Tris ions. The positively charged Tris ions migrate through the gel to the cathode where they neutralise hydroxide ions to form Tris molecules and water. Thus, the Tris-based buffering mechanism causes a constant pH in the buffer system. At 25 °C Tris buffer has an effective pH range between 7.5 and 9.0. Under the conditions given here the effective pH is shifted in the range of about 10.0 to 10.5. Native buffer systems all have low conductivity and range in pH from 3.8 to 10.2. Continuous native buffer systems are used to separate proteins according their pI.
Although the pH value 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 and isolated in different fractions. Provided that irreversible denaturation cannot be demonstrated, most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C. As the Joule heat and temperature generated during electrophoresis may exceed 50 °C, and thus, have a negative impact on the stability and migration behavior of proteins, the separation system, including the electrophoresis chamber and a fraction collector, is cooled in a refrigerator at 4 °C. Overheating of the gel is impeded by internal cooling of the gel column and by generating a constant power.
Gel properties and polymerization time
Best polymerization conditions for acrylamide gels are obtained at 25-30 °C and polymerization seems terminated after 20-30 min of reaction although residual monomers are detected after this time. The co-polymerization of acrylamide monomer/N,N'-Methylenebisacrylamide cross-linker initiated by ammonium persulfate /tetramethylethylenediamine reactions, is most efficient at alkaline pH. Thereby, acrylamide chains are created and cross-linked at a time. Due to the properties of the electrophoresis buffer the gel polymerization is conducted at pH 10.00 making sure an efficient use of TEMED and APS as catalysts of the polymerization reaction, and concurrently, suppressing a competitive hydrolysis of the produced acrylamide polymer network. Otherwise, proteins could be modified by reaction with unpolymerized monomers of acrylamide, forming covalent acrylamide adduction products that may result in multiple bands.Additionally, the time of polymerization of a gel may directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression and dilatation of the gels and their pores with the longer incubation times. In order to ensure maximum reproducibility in gel pore size and to obtain a fully polymerized and non-restrictive large pore gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature. The exothermic heat generated by the polymerization processes is dissipated constantly while the temperature may rise rapidly to over 75 °C in the first minutes, after which it falls slowly. After 69 hr the gel has reached room temperature, and thus, is in its lowest energy state because the chemical reactions and the gelation are terminated. Gelation means that the
solvent gets immobilized within the polymer network by means of hydrogen bonds and also van der Waals forces. As a result, the prepared gel is homogeneous, inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins. Sieving effects due to gravity-induced compression of the gel can be excluded for the same reasons. In a medium without molecular sieving properties a high-resolution can be expected.
Before an electrophoretic run is started the prepared 4% T, 2.67% C gel is pre-run to equilibrate it. It is essentially non-sieving and optimal for electrophoresis of proteins greater than or equal to 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 are negligibly low, and thus, the proteins separate cleanly and predictably at a polymerization time of 69 hr. The separated metalloproteins including biomolecules ranging from approximately < 1 ku to greater than 30 ku are not dissociated into apoproteins and metal cofactors.
Reproducibility and recovery
The bioactive structures of the isolated protein molecules do not undergo any significant conformational changes. Thus, active metal cofactor-containing proteins can be isolated reproducibly in the same fractions after a PAGE run. A shifting peak in the respective electropherogram may indicate that the standardized time of gel polymerization is not implemented in a PAGE experiment. A lower deviation of the standardized polymerization time stands for incomplete polymerization, and thus, for inherent instability due to gel softening during the cross-linking of polymers as the material reaches swelling equilibrium, whereas exceeding this time limit is an indicator of gel aging. The phenomenon of gel aging is closely connected to long-term viscosity decrease of aqueous polyacrylamide solutions and increased swelling of hydrogels.Under standard conditions, metalloproteins with different molecular mass ranges and isoelectric points have been recovered in biologically active form at a quantitative yield of more than 95%. By preparative SDS polyacrylamide gel electrophoresis standard proteins with molecular masses of 14-66 ku can be recovered with an average yield of about 73,6%. Preparative isotachophoresis is applied for isolating palladium-containing proteins with molecular masses of 362 ku and 158 ku.