Novex wedgewell tris glycine

Laemmli’s (1970 (link)) protocol was employed for SDS-PAGE with minor modifications. A gradient gel (4–20%; Novex™ WedgeWell™ Tris-Glycine, Thermo Fisher Scientific GmbH, Dreieich, Germany) was used to separate proteins. Broad range protein markers (10–200 kDa; P7719S, New England Biolabs GmbH, Frankfurt am Main, Germany) were utilized as reference proteins for molecular mass estimation. Protein bands were visualized by Coomassie staining using GelCode™ Blue Safe dye (Thermo Fisher Scientific GmbH, Dreieich, Germany).

In order to investigate the potential involvement of an endogenous K. phaffii endopeptidase with trypsin-like activity in the maturation of Tc-LysN zymogen into its active form, 250 μL of concentrated BMMY culture supernatant (post 24 h induction) was incubated with 425 USP-U of porcine pancreatic trypsin (Carl Roth, Karlsruhe, Germany) at pH 7.5 (60 mM MOPS) for 60 min at 37 °C. To account for any changes induced simply because of temperature and/or prolonged incubation time, two controls were also set-up: one with only culture supernatant without trypsin and another with only trypsin and no culture supernatant. Samples were withdrawn at specified time points to prepare for SDS-PAGE analysis and assessment of Tc-LysN’s activity. Samples for SDS-PAGE analysis were immediately mixed with reducing sample loading buffer and heated for 5 min at 90 °C before being loaded onto a 4–20% precast gradient gel (Novex™ WedgeWell™ Tris-Glycine, Thermo Fisher Scientific GmbH, Dreieich, Germany). Samples withdrawn for Tc-LysN’s activity measurement were first incubated with 10 mM phenylmethylsulfonyl fluoride (PMSF; Carl Roth, Karlsruhe, Germany) for 30 min at room temperature to inactivate trypsin. The experiment was additionally conducted with varied parameters such as trypsin dose, incubation time, and incubation temperature.