LmrV (20 μg) was analysed by SDS-PAGE (13.5%) under reducing conditions (β-mercaptoethanol and heating) according to the Laemmli method (Laemmli, 1970 (link)). The gel was stained with PlusOne Coomassie Blue PhastGel™ R-350 (GE Healthcare) and bands obtained were submitted to mass spectrometry analysis. A low range (6.5 – 66 kDa) molecular mass marker (Sigma-Aldrich, USA) was also loaded on the gel and the mass of hyaluronidase was estimated based on the migration pattern in the gel.
Additionally, SDS-PAGE (10%) containing hyaluronan (0.4 mg/mL) in gel matrix was carried out according to Cevallos et al. (1992) (link). Part of the gel was stained with PlusOne Coomassie Blue PhastGel™ R-350 (GE Healthcare) and the remaining with Stains-all (Sigma Chemical Co.) to detect hyaluronidase activity.
Additionally, SDS-PAGE (10%) containing hyaluronan (0.4 mg/mL) in gel matrix was carried out according to Cevallos et al. (1992) (link). Part of the gel was stained with PlusOne Coomassie Blue PhastGel™ R-350 (GE Healthcare) and the remaining with Stains-all (Sigma Chemical Co.) to detect hyaluronidase activity.