Pageruler unstained protein ladder

The dried fractions were redissolved in 10 µL reducing 2× SDS sample buffer, heated for 10 min at 95 °C, and separated using 12% SDS-PAGE (SurePage Bis-Tris, Genscript, Piscataway, NJ, USA) run with MES buffer at 200 V for 21 min. A PageRuler Unstained Protein Ladder (Thermo Scientific, Waltham, MA, USA) was used as the protein standard. Gels were short-washed with water three times. Proteins were fixed three times for 10 min each with hot fixation buffer (aqueous, 40% (v/v) methanol, 10% (v/v) acetic acid), stained for 45 min in hot fast staining buffer (aqueous, 0.3% (v/v) HCl 37%, 100 mg/L Coomassie 250G) under constant mild shaking, and kept overnight at 4 °C in storage buffer (aqueous, 20% (v/v) methanol, 10% (v/v) acetic acid) for destaining. The produced gels were then scanned for documentation and quantification.
To produce profiles that could allow the assessment of similarities and differences among the 49 individual venoms, 20 µg of each lyophilised venom sample was loaded in 10 µL reducing 2× SDS sample buffer and subjected to SDS-PAGE profiling following the same protocol applied for the venom pool. The resulting gels were scanned for documentation, and the obtained digital images were used for statistical analysis. SDS-PAGE profiling was performed once for each venom sample.

The purification of the recombinant proteins was carried out as described before (28 (link)). Briefly, the plasmids containing genes of interest fused to 6xHis-tags were transformed and expressed in E. coli BL21(DE3) strain and the recombinant proteins were later purified by metal ion affinity chromatography. The individual proteins PE18 (Rv1788), PE31 (Rv3478) and PPE26 (Rv1789) were purified by the on-column refolding method using an 8M Urea gradient. Purity was assessed by SDS-PAGE (>97% purity) and identity confirmed by Western blot. Molecular weight references for SDS-PAGE and Western blot were provided using PageRuler Unstained Protein Ladder (Thermofisher, 26614) and PageRuler Prestained Protein Ladder (Thermofisher, 26616), respectively. The concentration of the recombinant proteins was determined using the Lowry assay (BioRad Laboratories, Inc.) and the endotoxin content determined using Limulus amebocyte lysate (LAL) test (PYROTELL^®-T Lysate).

Skin mucus proteins were separated on 10% (v/v) polyacrylamide gels (acrylamide/N, N0-ethylene-bis-acrylamide, 200:1). A total of 10.3–20 µg for the sample of proteins in Laemmli buffer was boiled for 5 min at 100 °C and centrifuged at 10,000× g for 2 min at 4 °C. The samples were then separated per well in a Mini-PROTEAN 3 cell (Bio-Rad, Hercules, CA, USA). A PageRuler unstained protein ladder was also used as the molecular weight (MW) indicator (Thermo Fisher Scientific, San Jose, CA, USA). Running conditions were 80 V for the first 20 min and then 150 V until the end of the electrophoresis. Gels were stained overnight with Coomassie Brilliant Blue R-250 (VWR International, Radnor, PA, USA), unstained by using a solution composed of 25% ethanol and 8% acetic acid, washed with 50% methanol (v/v), and scanned at 200 dpi.

A standard PURE reaction programmed with 17.5 nM (250 ng) of a linear Rep DNA template (under control of a T7 promoter) was supplemented with 0.6 µl FluoroTect™ Green_Lys tRNA (FluoroTect™ Green_Lysin vitro translation labelling system, Promega). Template DNA was omitted in the negative control reaction. Samples were incubated for 2  h at 37 °C in a nuclease-free PCR tube (Thermo Fisher Scientific) using a ProFlex PCR System (Thermo Fisher Scientific) and subsequently treated with 0.6 μl RNase Cocktail™ Enzyme Mix (0.5 U/μl RNase A and 20 U/μl RNase T1, Thermo Fisher Scientific) for 15 min at 37°C to degrade non-incorporated Green_Lys tRNA. 7.5 μl sample were mixed with an equal volume 2× Laemmli sample loading buffer (incl. 200 mM DTT) and denatured for 2.5 min at 65°C. Samples were analysed by conventional discontinuous SDS-PAGE (10% gel) run at 4°C (100 V for 10 min, then 200 V) on a Midi-format electrophoresis system (Atto). The fluorescent signal of the de novo expressed rep β subunit was imaged on a fluorescence laser scanner (Typhoon FLA 9000, GE Healthcare) at either 473 nm (blue LD laser/510LP filter) or at 532 nm (green SHG laser/575LP filter). Total protein and the molecular-weight size marker (PageRuler™ Unstained Protein Ladder, Thermo Fisher Scientific) were visualized after SYPRO Ruby (Bio-Rad) staining using the same instrument (473 nm, blue LD laser/575LP filter).

Purified PA-SR01 was diluted in SDS buffer (5:1, vol/vol) and heated at 95°C for 5 min. The sample was then resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using a Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories). The Mini-PROTEAN TGX stain-free precast gel was run in an SDS running buffer (pH 8.3) at 120 V for 1.5 h using a PageRuler unstained protein ladder (Thermo Fisher) for size calibration.

Protein concentrations were quantified using Bradford protein assay (Carl Roth, Germany) following TCA/acetone precipitation. Bovine serum albumin (BSA) was chosen for modeling standard curves and measurements were recorded in triplicates in 96-well plates on EnSpire® Multimode Plate Reader (PerkinElmer, USA). Protein extracts were conveyed on 12% one-dimensional SDS polyacrylamide gel electrophoresis, using Bio-Rad Mini-Protean II Equipment and PageRuler ™ Unstained Protein Ladder (ThermoFischer Scientific, USA), to assess the gross qualitative variances in protein profiles. After electrophoresis, gels were stained with Coomassie brilliant blue (CBB) R-250.