2.6.2. Gel Filtration Column Chromatography and Gel Electrophoresis (SDS–PAGE)

The PPPE extract was then purified by passing through a Tris-buffer (pH 7)equilibrated Sephadex G-50 column (Sigma-Aldrich). The PPPE was eluted using the same buffer, and 3 mL fractions (about 25 in number) at 1 mL min⁻¹ flow rate were collected, and their absorbance was read at 280 nm. Those fractions that exhibited maximum protease activity were pooled, concentrated, and kept for subsequent analysis.

Gel electrophoretic analysis was executed according to Laemmli [46], and the approximate molecular weight of the protease from strain HM48 was evaluated against a standard protein ladder (Spectra, Thermo Scientific).