Coomassie brilliant blue g 250

Graham flour was kindly donated by the bakery Hlebček d.o.o. (Pragersko, Slovenia). Analyses were conducted using carbon dioxide (99.5% purity) produced by Messer, Ruše. Ethanol, phosphoric acid, and Coomassie-Brilliant Blue G250 were supplied from Merck (Darmstadt, Germany), while bovine serum albumin (BSA), sodium acetate, potassium phosphate, l-3,4-dihydroxyphenylalanine, l-ascorbic acid, and ethylenediaminetetraacetic acid were supplied from Sigma (Steinheim, Germany). All other chemicals used in the laboratory were of analytical grade.

Sodium hyaluronate (HA, 1.0–1.8 MDa) was obtained from Lifecore Biomedical (Chaska, MN, USA). BSS was obtained from Sigma Aldrich (Gillingham, Dorset, UK). SO (S500 and S1000) was purchased from VWR International Ltd (Lutterworth, Leicestershire, UK). Coomassie^® Brilliant Blue G250 (Merck, Darmstadt, Germany) was used for visualization.

The presence of putative bacteriocin was checked by running the CFS and the precipitated sample in SDS-PAGE [24 (link)]. Here, Tris-glycine sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used, employing a vertical slab gel apparatus (MiniPROTEAN Tetra Cell, BioRad, Hercules, CA, USA) with 4% stacking gel and 12% separating gel. SDS-PAGE was run at 100 V for 1.5 h along with the Spectra™ Multicolor Broad Range Protein Ladder (10 to 260 kDa, Thermo Fisher Scientific). Upon completion, the gel was stained with Coomassie brilliant blue G-250 (Merck, Darmstadt, Germany) and destained using a methanol and acetic acid solution with a 3:1 ratio. The proteins observed below ~10 kDa of the ladder were putatively identified as bacteriocin.

The Sf9 cells were infected with virus rAc-tVP2 or rAcBac at an MOI of 5.0 and cultured at 27 °C for 96 h. The cells were collected via centrifugation, resuspended in PBS (pH 7.4), and lysed by ultrasonic crushing. The cell debris and supernatant were separated via centrifugation at 4 °C with 12,000 rpm for 30 min, and the total protein was boiled for 10 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, Shanghai, China) and subjected to SDS-PAGE. Samples on 10% SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue G-250 or transferred onto nitrocellulose membranes (Merck Millipore, Burlington, MA, USA) using a Mini PROTEAN ^® Tetra Cell (Bio-Rad, Hercules, CA, USA). Mouse antiserum against the Flag tag (diluted 1:2000 in PBST, BOSTER, Wuhan, China) was used as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG (diluted 1:2000 in PBST, BOSTER, Wuhan, China) as the secondary antibody for western blot analysis. Antibody binding was detected using the enhanced chemiluminescence system (Merck, Darmstadt, Germany).

Calcitermin (Cal) was purchased from KareBay Biochem (Monmouth Junction, NJ, USA) with a certified purity of 98%. The copolymer poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) poloxamer 407 (p-407) (PEO98-POP67-PEO98), xanthan gum from Xanthomonas campestris (x-gum) composed of a β-(1→4)-D-glucopyranose glucan backbone with side chains of (1→3)-α-D-mannopyranose-(2→1)-β-D-glucuronic acid-(4→1)-β-D-mannopyranose on alternating residues, average molecular weight of about 2000 kDa, and Coomassie brilliant blue G 250 were purchased from Merck (Milan, Italy). Nylon membranes (2.5 cm diameter, pore size 0.2 μm) and nylon syringe filters (0.22 μm pores) were purchased from Merck (Milan, Italy). Solvents were of HPLC grade, and all other chemicals were of analytical grade.