Mini protein vertical slab electrophoresis system

SDS-PAGE was performed following the methods previously described by Laemmli (1970) [57 (link)] with modifications using a mini-protein vertical slab electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). Samples were dispersed separately in 5% SDS solution. The mixtures were incubated at 85 °C for 1 h and centrifuged at 8500× g for 5 min to remove insoluble substances. The dispersed samples were mixed with sample buffer (0.5 M Tris-HCl, pH 6.8) containing 5% SDS (w/v) and 20% glycerol (v/v) at a 1:1 (v/v) ratio. Samples were loaded onto polyacrylamide gels (8% separating matrix, 3% stacking matrix) and electrophoretically separated under a constant current of 20 mA/gel. The gel was then treated with 25% methanol (v/v) and 5% acetic acid (v/v) for 30 min and stained for 30 min with 0.1% Coomassie Brilliant Blue R-250 dye (w/v) (Bio-Rad Laboratories, Hercules, CA, USA) in 30% methanol (v/v) and 10% acetic acid (v/v), followed by 30 min of de-staining in 30% (v/v) methanol and 10% (v/v) acetic acid. The staining ratio of α1 to α2 were analyzed using Quantity One 4.6.0 (Bio-Rad Laboratories, Hercules, CA, USA). High-molecular-weight markers were used to estimate the molecular weight of the samples.

The SDS–PAGE of the sample was determined according to Laemmli’s method (1970) [47 (link)], with slight modifications. The lyophilised samples were dissolved in ultrapure water at 0.1% (w/v). The resulting solution was mixed with a buffer (0.025 M Tris-HCl, pH 8.3, including 0.192 M glycine and 0.1% w/w SDS) at a ratio of 1:4 and then denatured at 100 °C for 3 min. SDS–PAGE of the samples was determined on a mini protein vertical slab electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). Then, 6 μL of sample solution and marker (26634) was loaded on polyacrylamide gels consisting of 8% separating gel and 3% stacking gel. Electrophoresis was performed at a constant voltage of 110 V until the end of the separation. The gels were removed, fixed, stained and destained separately for 30 min.