QPNC-PAGE

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 the ability to isolate active proteins in biological samples for more detailed examination of chemical structure and physiological function.

Many of these cofactors (e.g., iron, copper, or zinc) play a key role in vital enzymatic catalytic processes or stabilize globular protein molecules driven by protein-cofactor interactions.

[9] Here, a similar mechanism is accomplished in a commercially available electrophoresis chamber for separating charged biomolecules, for example, superoxide dismutase (SOD)[10] or allergens,[11] at constant pH conditions and different velocities of migration depending on different isoelectric points of zwitterions.

[12] A shifting peak in the respective electropherogram indicates that the standardized time of gel polymerization (69 hr, RT) is not implemented in a PAGE experiment.

[30] The phenomenon of gel aging is closely connected to long-term viscosity decrease of aqueous polyacrylamide solutions[31] and increased swelling of hydrogels.

[32] 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%.

[19] By preparative SDS-PAGE standard proteins (cytochrome c, aldolase, ovalbumin and bovine serum albumin) with molecular masses of 14–66 ku can be recovered with an average yield of about 73.6%.

[34] Physiological 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 in an aliquot of a fraction by inductively coupled plasma mass spectrometry (ICP-MS)[35] or total reflection X-ray fluorescence (TXRF),[36] for example.

[37][38] Another established high sensitive detection method for the determination of trace elements in biological samples is graphite furnace atomic absorption spectrometry (GF-AAS).

[39] 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 a few specific PAGE fractions, the related structures of these bioactive analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions.

[41] Active CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ion species (e.g., Cu1+/2+, Zn2+, Fe2+/3+, Mn2+, Ni3+) in organisms, and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm.

[42] Otherwise, free or loosely bound transition metal ions take part in Fenton-like reactions in which deleterious hydroxyl radical is formed, which unrestrained would be destructive to proteins.

[46] (Q)PNC-PAGE was originally developed at Research Centre Jülich in the 1990s to optimize the chemical composition and physical properties of the acrylamide gel matrix.

Folding funnel : By minimizing its free energy an unfolded poly peptide chain assumes its native structure. In order to protect the native state from unfolding, the run conditions for native gel electrophoresis must be precisely adapted in terms of downstream analysis of native proteins . [ 1 ]
Equipment for bioanalytical continuous- elution gel electrophoresis: electrophoresis chamber, peristaltic pump , fraction collector, buffer recirculation pump and UV detector (in a refrigerator), power supply and recorder (on a table). [ 7 ]
Electropherogram showing four PAGE runs of a high molecular weight plant protein (≈ 200 k Da ) as a function of time of polymerization of the gel. Detection method for determination of Cd cofactor concentrations (in μg/L): GF-AAS . [ 7 ]