Colloidal coomassie brilliant blue g 250

The proteins in the venom samples were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 4–12% Criterion XT gradient gels (Bio-Rad) with XT MES running buffer at 125 V for 1.5 h, alongside protein molecular weight markers. The gels were stained for 1.5 h using a 1 : 1 mixture of Coomassie Brilliant Blue R-250 and colloidal Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific), washed in Millipore water overnight and then scanned. For liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis, protein bands from each gel lane were excised and digested with trypsin [41 (link)]. Further details of LC–MS sample processing, data acquisition and data processing are presented in electronic supplementary material, Methods S1, section 1.

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4–12% Criterion XT gradient gels (BioRad, Hercules, CA, USA) with XT MES running buffer (BioRad, Hercules, CA, USA). Gels were run at 125 V for 1 h along with a pre-stained protein standard (Thermo Fisher Scientific, Waltham, MA, USA) and stained for 1.5 h in a 1:1 (v/v) mixture of Coomassie Brilliant Blue R-250 and colloidal Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific, Waltham, MA, USA). After washing in Millipore water overnight to remove excess dye, the stained gels were scanned and analyzed. From selected gel lanes, the protein bands were excised for tryptic digestion [38 (link)] and reconstituted in 50 μL of 1% formic acid in water. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and subsequent data collection were performed as described previously [7 (link)].

The venom proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) on 4%–12% Criterion XT gradient gels (BioRad) with XT MES running buffer at 125 V for 1.5 hr. Prestained and unstained high‐mass‐precision protein markers were used to determine the molecular weight (kDa) of the venom proteins. Gels were stained with a 1:1 mixture of Coomassie Brilliant Blue R‐250 and colloidal Coomassie Brilliant Blue G‐250 (Thermo Fisher Scientific) for 1.5 hr. Excess dye was removed by washing in Millipore water overnight, and the stained gel was then scanned and analyzed.
For LC‐MS/MS analysis, protein bands from each gel lane were excised as 29 molecular weight fractions for tryptic digestion (Shevchenko, Tomas, Havli, Olsen, & Mann, 2006). Further details of LC‐MS sample processing, data acquisition and data processing, such as search parameters specifying mass measurement accuracy, minimum number of product ion matches per peptide, minimum number of product ion matches per protein, minimum number of peptide matches, and maximum number of missed tryptic cleavage sites can be found in Methods S1, Section 1.