4.9. Native Polyacrylamide Gel Electrophoresis (PAGE)

The detection of the isoenzyme patterns for two different groups of enzymes was performed on a native poly-acrylamide gel electrophoresis in the presence of low amounts of SDS (0.1%) in the running buffer. No reducing agents were present [54]. For the detection of the enzyme activities, the electrophoresis was carried out at 4 °C. Total proteins were separated and the resulting patterns were stained for peroxidase (POX) and superoxide dismutase (SOD) activities and total protein (Coomassie). Equal amounts of protein (50 μg) were loaded in each lane of the gels. The resolving gel and stacking gel had acrylamide concentrations of 10% and 5%, respectively. A molecular mass marker (Thermo Fisher Scientific, Dreieich, Germany) was run on all gels along with the tested samples.

The detection of the peroxidase isoenzyme patterns was performed according to Ludwig-Müller et al. [54] using benzidine-guaiacol as substrates to visualize the bands. The staining solution comprised of solution A: 40 mL 0.2 M Na-acetate, 4 mL 5 mM MnSO₄, 4 mL 0.35% H₂O₂ and solution B: 20 mg benzidine (4,4′-diaminobiphenyl), 10 mL 10% acetic acid, 54 μL guaiacol. When the run of the polyacrylamide gel was completed, the gel was quickly rinsed in H₂O and then placed into solution A. Incubation was performed at room temperature. Under constant shaking, solution B was added to the gel. Staining was continued until the POX bands were visible on the gel. The reaction was stopped by replacing the staining solution several times with water. The density of the bands was detected and displayed using the GelEval software free trial version 1.35 (FrogDanceSoftware, Cambridge, UK).

SOD isoenzymes were detected in the gels by their ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). The staining of the gels was performed based on the method described by Pitzschke et al. [55] with some modifications. The gels were rinsed in cold distilled water, then incubated for 20–25 min in 2 mM NBT (made in 100 mM potassium phosphate buffer, pH 7.8) under constant agitation in light at 4 °C. The NBT solution was then replaced with riboflavin solution [45] (0.030 mM riboflavin, 1% N, N, N′, N′-tetramethylethylenediamine (TEMED) in the same buffer) and the gels were further incubated for 25–30 min at 4 °C in darkness. Finally, the gels were briefly rinsed in distilled water and SOD activity was shown by white bands against a violet background.

The total protein pattern was determined using the colloidal Coomassie staining method based on the protocol of Neuhoff et al. [91] with some modifications. The gel was incubated for 20 min in 50 mL solution A (10% (w/v) ammonium sulfate, 2% phosphoric acid in distilled H₂O) containing 1.25 mL solution B (5% (w/v) Coomassie Brilliant Blue G250 in distilled H₂O) under continuous shaking. The gel was then destained in 25% methanol in distilled H₂O followed by destaining in methanol until the background was clear.