Mini protean 2 electrophoresis cell

SDS-PAGE was carried out according to Laemmli’s method^{58 (link)} using a Mini-Protean II electrophoresis cell (BioRad, Marnes-la-Coquette, France).

Whole cell lysates were prepared using the RIPA lysis buffer supplemented with protease inhibitors. HL-60 and ML-1 cells were washed with ice-cold PBS, lysed on ice for 15 min and cleared by centrifugation at 15,000× g at 4 °C for 15 min. The protein content was determined via Bradford protein assay using bovine serum albumin (BSA) as a standard. Proteins (30 μg from each experimental group) were separated by Mini-PROTEAN^® TGX™ gels (Cat. No. 456–1093; BioRad, Hercules, CA, USA) using SDS-PAGE (BioRad Mini-PROTEAN II Electrophoresis Cell), followed by transferring them to PVDF membranes. The membranes were blocked in 5% BSA/TBST and probed with anti-MCL-1 primary antibody (1:1000 in blocking buffer) at 4 °C overnight. Next, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody diluted to 1:1000. The bands were detected by chemiluminescence using the WesternBright Sirius Western blotting HRP substrate (Advansta, Menlo Park, CA, USA), visualized using a ChemiDoc-It Imaging System (UVP, LLC, Cambridge, UK) and quantified using ImageJ analysis software (US National Institute of Health, Bethesda, MD, USA). The blots were then stripped and probed for anti-β-actin used as an internal control for the samples.

Protein samples were fractionated by SDS-PAGE on 12% running gels, with 5% stacking gels. Electrophoresis was performed in a BioRad Mini-Protean II electrophoresis cell for 1 h, at 200 volts, constant voltage. Gels were visualized by staining with Coomassie Blue G-250 dye. Supplementary Figure 3 shows SDS-PAGE of purified recombinant OsDHPD-T, OsDHP-T2, and Osß-UP proteins.

For protein determination, 15 μL aliquots of diluted honey samples (50% w/w in distilled water) were loaded on 12% SDS-PAGE gels and separated using a Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA). Protein content was assessed after gel staining with Coomassie Brilliant Blue R-250 (Sigma-Aldrich, Darmstadt, Germany) or Serva Blue (Serva, Heidelberg, Germany).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of SABU was conducted under reducing and non-reducing conditions using the MiniPROTEAN II Electrophoresis Cell (BioRad, USA) as adapted from Studier34 (link). Bio-Rad broad-range prestained SDS-PAGE standards (6.5–200 kDa) and 15 μL SABU (3 mg/ml) were loaded onto 12.5% gel and the electroporesis running condition was standardized as per previously reported35 (link). Fast protein liquid chromatography (FPLC) of the antivenom (approximated to 1 mg in 100 μL injection volume) was performed using a Superdex 200 HR 10/30, 13 μm SEC 10 × 300 mm (GE Healthcare, Sweden). Elution buffer was 100 mM sodium phosphate, 0.15 M NaCl, pH 7.4 at a flow rate of 0.5 ml/min. Proteins in the antivenom were detected by absorbance readings at 280 nm for 30 min. The column was calibrated using the following protein standards supplied by Bio-Rad (Bio-Rad Gel filtration Standard): thyroglobulin (670 kDa), α-globulin (158 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa).

In order to identify the antibiofilm components of ES products, SPE eluate was fractionated using a reverse phase (RP)-HPLC chromatography (Beckman System Gold, USA) coupled with a C18 column (250 mm × 4.6 mm; 5 μm) (Grace, USA) at a flow rate 0.3 mL/min, by using a gradient from 0 to 90% (v/v) acetonitrile (containing 0.1% (v/v) trifluoroacetic acid), during 70 min. The fractions were lyophilized and dissolved in 100 μL of TSB and tested for antibiofilm activity against S. aureus. The active fraction was then fractionated using a C4 column (250 mm × 4.6 mm; 5 μm) (Grace, USA) and tested under the same conditions. The purity of active fractions was checked by electrophoresis on 16.5% Tricine-SDS-PAGE gel using a Mini-Protean II electrophoresis cell (Bio-Rad, CA, USA). Protein bands were detected after staining with Coomassie Brilliant Blue R-250.