Broad range molecular weight standard

Protein separation by SDS-PAGE was performed following the method described by Laemmli^{37 (link)} using a Mini Protean II system (Bio-Rad, 2000, CA, USA). Samples were mixed with 4 × sample buffer containing 50 mM Tris–HCl pH 6.8, 20% glycerol, 10% SDS, 10% β-mercaptoethanol and 0.05% bromophenol blue. The samples were then boiled for 10 min and loaded into either 10% or 15% polyacrylamide gel. A broad-range molecular weight standard (Bio-Rad, 1610317, CA, USA) was also loaded into the gel. Electrophoresis was carried out at 90-V and room temperature using a vertical electrophoresis unit (Bio-Rad Protean II, CA, USA). After electrophoresis, the gels were stained with Coomassie blue solution (0.05% (w/v) Coomassie Brilliant Blue R-250, 7% v/v acetic acid, 40% v/v methanol) for 2 h. Protein bands were then visualized by soaking the gels in a distaining solution (7% v/v acetic acid and 40% v/v methanol) for 2 h. Gel images were digitalized using a gel imager (Chemi Doc XRS, Bio-Rad, CA, USA). The relative molecular mass of protein bands was determined by comparing their migration with the molecular weight standard.

PAGE was performed in a 6% polyacrylamide gel (37.5% acrylamide/1% bisacrylamide) using a mini-protean system (Bio-Rad, Mexico City, Mexico). Briefly, GAGs in Tris-HCL buffer (pH 6.8) were mixed with non-reducing sample buffer, boiled for 5 min, loaded onto the gel and electrophoresed at 100 V for 110 min. The gel was stained with 0.5% Alcian Blue solution in 3% acetic acid/25% isopropanol for two hours and de-stained with a 10% acetic acid/40% ethanol solution overnight. Finally, the gel was digitized. A broad range molecular weight standard (Bio-Rad, Mexico City, Mexico) was used.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli^{75 (link)}. One milligram of lyophilized ASM and AIM of the shell from P. mazatlanica were mixed with 50 µL of 4 × sample buffer (05 M Tris–HCl pH 6.8, 20% glycerol, 10% SDS, 10% β-mercaptoethanol and 0.05% bromophenol blue) respectively. Samples were boiled for 10 min and then centrifuged 1 min at 12,000 × g. The supernatant (20 µL) was loaded into a 12% polyacrylamide gel. Broad range molecular weight standard (Bio-Rad, 1610317, California, USA) was loaded into the gel. Electrophoresis was run at 90-V at room temperature, using a vertical electrophoresis unit (Bio-Rad Protean II, California, USA). After electrophoresis, the gels were stained for 2 h with staining solution (0.05% (w/v) Coomassie Brilliant Blue R-250, 7% (v/v) acetic acid, 40% (v/v) methanol). Proteins were revealed by soaking for 2 h in distaining solution (7% (v/v) acetic acid and 40% (v/v) methanol). Gel proteins were analyzed using a gel imager (Chemi Doc XRS, Bio-Rad, California, USA).