Precision plus protein dual colour standard

Protein content was determined according to Bradford. A calibration curve was made with bovine serum albumin (BSA) in concentrations of 0.25–1.0 mg mL^-1. Enzyme purity was confirmed by SDS-PAGE under reducing conditions on a Mini-protean II system (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. Commercially prepared mini-protean TGX gels (Bio-Rad) were used with Coomassie InstantBlue (Expedion, Cambridge, UK), with the marker Precision Plus Protein^™ dual colour standards (Bio-Rad). Samples (8 μL) were loaded onto a gel, and the separation was done by applying 200 V for 45 min.

Supernatant was collected from cell culture on functionalised coverslips stored at − 80 °C prior to zymogram analysis. Supernatant was then mixed 1:1 with zymogram sample buffer (loading buffer, BioRad, UK) and 20 µL was loaded into each well of precast gelatin and casein gels (BioRad, UK). Precision Plus Protein ™ Dual Colour Standards (BioRad, UK) were used as molecular standards (10 µL loaded per gel). The gels were run at 200 V in running buffer (10% Tris/Glycine/SDS running buffer diluted PBS) for 60 min in the Criterion™ gels until the bands reached the bottom. Gels were soaked in zymogram renature buffer (10% renature buffer in PBS, BioRad UK) using gentle agitation for 45 min with buffer changes at 15 and 30 min. The gels were then incubated in zymogram development buffer (10% development buffer in PBS) overnight at 37 °C. Afterwards, gels were stained in 0.5% (w/v) Coomassie Brilliant Blue R-250 in 4% methanol 10% acetic acid at room temperature for 60 min with gentle agitation. The Coomassie Blue solution was replaced with destain solution (4% methanol/10% acetic acid in PBS), which was replaced with 3 changes every 15 min (at room temperature) until bands were visible. Gels were imaged using a fusion Fx, Vilber Lourmat and bands were quantified using Fiji software (ImageJ derivative, free download from NIH).

The scale wastes of parrotfish (Scarus sordidus Forsskål, 1775) were provided by a fishmonger in the Kota Kinabalu fish market (Sabah, Malaysia). All fish scales (approximately five kilograms) were packed in a polyethylene bag containing a 2 : 1 (g/g) ratio of ice to scale, and the packed samples were then transported to the laboratory. Upon arrival, the scales of parrotfish were rinsed with running tap water and dried in an electric cabinet dryer (WS340, Tsung Hsing, Kaohsiung city, Taiwan). Acetic acid (100056), Folin–Ciocalteu's phenol reagent (109001), N, N, N′,N′-tetramethyl ethylene diamine (TEMED) (110732), Coomassie Blue R-250 (112553), acrylamide (800830), and sodium dodecyl sulfate (SDS) (817034) were supplied from Merck (Darmstadt, Germany). Lowry reagent (L1013), Trizma® hydrochloride (T3253) and bovine serum albumin (BSA) (A3733) were obtained from Sigma Chemical Co., (St. Louis, USA). Precision plus protein dual colour standards (molecular weight markers) (1610374) were supplied by Bio-Rad Laboratories (Hercules, CA, USA). Other reagents and chemicals used in the present study were of analytical grade.

30 μg protein were loaded into pre-cast 4–15% Mini-PROTEAN^® TGXTM gels (Bio-Rad) alongside Precision Plus Protein™ Dual Colour Standards (Bio-Rad) and run at 150V in Tris/Glycine/SDS Running Buffer (Bio-Rad). The Trans-Blot^® Turbo™ Transfer Starter System, Mini PVDF (Bio-Rad) was used to transfer protein to membranes followed by blocking for 1 h, overnight incubation with primary antibodies at 4°C and 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at RT. ClarityTM ECL Western Blotting Substrate (Bio-Rad) and ChemiDoc Touch (Bio-Rad) were used to image the membranes. Relative protein band intensities were quantified and normalized against Vinculin using densitometric analysis.

Fluorescein-cadaverine (FC, Molecular Probes^TM A10466) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Putrescin, 4’,6-diamidino-2-phenylindole (DAPI), TGase assay kit (CS1070-1KT), RPMI 1640 medium, and goat serum were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Diethyl aminoethyl (DEAE) and Heparin Sepharose were purchased from Cytiva (Marlborough, MA, USA) and Sigma-Aldrich, respectively, and nitrocellulose membrane (Amersham Protran Supported) was purchased from GE Healthcare (Chicago, IL, USA). Human TGase 2 rabbit polyclonal antibodies (orb2986) were purchased from Biorbyt (Cambridge, UK). Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Novus Biologicals Europe, UK. FITC-tagged anti-rabbit antibodies were obtained from Chemicon (Temecula, CA, USA). Fetal bovine serum (FBS, South America origin, EU Approved) was obtained from EuroClone, Italy. Protease inhibitor cocktail and the Western Blot detection system SuperSignal™ West Femto Maximum Sensitivity Substrate were obtained from Thermo Scientific (Waltham, MA, USA). Protein molecular mass markers ECL Plex Fluorescent Rainbow and Precision Plus Protein™ Dual Colour Standards were obtained from BIO-RAD (Hercules, CA, USA). All other chemicals were obtained from Sigma-Aldrich.

The analyses of the protein extracts were achieved by mixing each sample individually with 6× protein sample buffer and heated to 95 °C for 5 min. The proteins were separated on commercial 4–20% gradient TGX gels (Bio-Rad, Hercules CA, USA). The molecular weight markers used for stained gels were Precision Plus Protein™ Dual Colour Standards (BioRad). The separated protein bands were visualized in a Gel Doc™ EZ Imager (BioRad). The proteins were electrophoretically transferred from gel to PVDF membranes. Prior to the transference, the membranes were blocked for 2 h at room temperature (RT) with 5% non-fat dry milk in PBST (phosphate-buffered saline, 0.05% Tween-20) followed by the incubation with the first antibody, goat IgG anti-beta conglutin (dilution 1:2000), overnight at 4 °C in continuous agitation. After washing 5 times with PBST, the membrane was incubated with secondary antibody goat anti-IgG rabbit conjugated with horseradish peroxidase (dilution 1:10,000) in 2% non-fat dry milk in PBST for 2 h at RT. The membrane was washed 5 times with PBST and the chemiluminescence signal was developed by membrane incubation with ECL Plus chemiluminescence substrate following the manufacturer’s instructions (BioRad). The light signal was detected by exposure of the membrane to C-Digit Blot Scanner (LI-COR).