Precision plus protein dual xtra standard

Twenty-five μg of each venom were loaded on 12.5% SDS-PAGE gels under reducing conditions. Samples were diluted using Sample buffer 5X (10% Glycerol, 2.5% SDS, Tris-HCl 50 mM pH 6.8, 5% 2-mercaptoethanol, 0.002% bromophenol blue) to a final volume of 20 μL and boiled for 5 min. Electrophoresis was performed with a constant voltage of 80 V for 15 min and then 100 V for approximately 60 min. Gels were stained with G-250 Coomassie Brilliant Blue. Apparent molecular weights were determined comparing migration distance with 5 μL of molecular weight markers (Precision Plus Protein Dual Xtra Standards, Bio-Rad) using ImageJ software version 1.50i.

Briefly, 5 μg of each antibody sample (commercial α-ToxAb, Bz-DFO-ToxAb, and [⁸⁹Zr]Zr-DFO-ToxAb) was mixed with loading buffer under reducing conditions to preserve antibody integrity. Mixtures were boiled for 6.5 min at 95°C and then loaded onto a 7.5% sodium dodecyl sulfate-polyacrylamide gel along with standard molecular weight markers (Precision Plus Protein Dual Xtra Standards; Bio-Rad, Hercules, CA, USA). Samples were run at 90 V for 2.5 h before staining the gel with Coomassie Blue (Bio-Rad, Hercules, CA, USA) for 45 min. The gel was destained with a solution of 50% methanol, 40% acetic acid, and 10% water for 24 h.

Proteins were separated on a polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) using 3% thickening silica gel and 12% separating gel. Protein was denatured in a temperature range of 90–100°C for 5 min with concurrent shaking (Eppendorf Thermomixer Comfort, Germany). Then, electrophoresis was performed using a Mini Protean® 3 Bio-Rad device at constant current of 20 mA (voltage is 200 V) in 1x Tris-glycine buffer with a pH of 8.3. An appropriate molecular weight marker (Precision Plus Protein Dual Xtra Standards, Bio-Rad) was used to determine the mass of protein fractions. For visualization, the gel was stained with Coomassie Brilliant Blue R-250. Electrophoretically separated proteins were documented using a gel recording system (GelDoc 2000, Bio-Rad, France). Proteins were analyzed using the Quantity One version 4.2.1 (France) computer program [38 (link)].

The protein fractions were resolved on 10% polyacrylamide gels under denaturing conditions (SDS-PAGE). Protein visualization was performed by Coomassie blue R-250 and silver nitrate staining. The molecular mass of the monomeric subunit of myrosinase was determined as a function of its migration distance, using the Precision Plus Protein Dual Xtra Standards (Bio-Rad, Hercules, CA, USA) protein markers.
For Western Blot analysis, the proteins resolved by SDS-PAGE were transferred to a nitrocellulose membrane (pore size 0.25 µm), using an electro blot chamber (Mini Trans-Blot^® Module, Bio-Rad, Hercules CA, USA). The membrane was incubated in a blocking solution containing 5% skim milk in TBS-T buffer (20 mM Tris·HCl pH 7, 0.1 M NaCl, 0.05% Tween-20), for 1 h at room temperature. The membrane was incubated for 2 h at room temperature and under constant shaking with the primary anti-His antibody conjugated to the HRP enzyme (1:1000) to detect the recombinant protein produced in E. coli BL21(DE3)-myr (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and the primary anti-GST antibody conjugated to the enzyme HRP (1:1000) to detect the recombinant protein produced in S. cerevisiae MGY70-myr. The development was carried out using autoradiography films and the WESTAR substrate (Cyanagen, Bologna, Italy).

The supernatants of the protein solutions after the heat treatment were dissolved in 62.5 mM Tris–HCl (pH 6.8) loading buffer containing 2 % (w/v) SDS, 5 % sucrose, 5 % β-mercaptoethanol, and 0.01 % bromophenol blue. The samples were heated for 5 min in boiling water and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 5–20 % gradient gel (e-PAGEL, ATTO Co., Tokyo, Japan) with a molecular weight marker (Precision Plus Protein Dual Xtra Standards; BIO-RAD, Hercules, CA, USA). The gels were then stained using silver nitrate.

SDS-PAGE was performed using the small size 10% acrylamide gels (8.3 cm × 7.3 cm), 1 mm thick, which was casted with 30% Acrylamide/Bis solution (37.5:1). The protein samples (20 μl) were loaded into the gel well. As a standard, the Precision Plus Protein™ Dual Xtra Standards from Bio-Rad was used. Then, electrophoresis began with an initial voltage of 30 V and maintain at this voltage until the sample has completely entered the gel. Then the electrophoresis was carried out at a constant voltage of 200 V for 2 h. Gels were stained with Bio-Safe™ Coomassie G250 Stain. The sample lane of the gel was separated into two fractions at position of about 50 kD for liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis.