Casein

Protease activity was measured by modified Lowry method (Lowry et al., 1951 (link); Boominadhan et al., 2009 ) whereas protein concentration was estimated by Bradford method (Bradford, 1976 (link)). Reaction mixture containing 1% casein (Sisco Research Laboratories Pvt. Ltd., India, www.srlchem.com) as a substrate (400 μl) in 0.1M potassium chloride–sodium hydroxide (pH 12) and purified enzyme (200 μl) was incubated for half an hour at 37°C. The reaction was terminated by addition of 600 μl of 10% trichloroacetic acid (Sisco Research Laboratories Pvt. Ltd., India, www.srlchem.com) followed by incubation for 10 min at room temperature. The reaction mixture was centrifuged at 10,000 g. Supernatant (200 μl) was added to 0.4M sodium carbonate (800 μl) and 1N Folin phenol Ciocalteau reagent (50 μl), incubated for half an hour at room temperature, and the absorbance measured at 660 nm. One unit enzyme activity was defined as the amount of enzyme that is needed to release 1 μg tyrosine per ml per minute under standard assay conditions.

Bovine serum albumin, bovine α-trypsin, α-chymotrypsin, casein and Polyvinylpyrrolidone (PVP) were procured from Sisco Research Laboratory, Mumbai, India. CNBr activated Sepharose 4B, Sephadex G-50 fine, BAPNA, GLUPHEPA, soybean trypsin-CI (soybean BBI), tricine, gelatin, phenylmethylsulfonyl fluoride (PMSF), Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), Np-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), Trizol reagent and Coomassie brilliant blue R-250 were purchased from Sigma Aldrich, United States. Immobiline pH gradient dry strips (IPG strips), IPG buffer, DTT, IDA, urea, thiourea, 3-[(3-Cholamidopropyl) dimethylammonio]-1-Propanesulfonate hydrate (CHAPS), CM5 sensor chips, amine coupling kit and HBS EP + 10X buffer were procured from GE Healthcare Biosciences Corp., United States. Verso cDNA synthesis kit, 50 bp DNA ladder, bicinchoninic acid (BCA) protein estimation kit, protein molecular mass standard, and 3 kDa cut-off SnakeSkin dialysis membrane were purchased from Thermo Fisher Scientific, United States. SYBR Green PCR Master Mix purchased from Takara Bio, Shiga, Japan and all other PCR components from New England Biolabs. Amicon ultra centrifugal filter units were purchased from Millipore Corporation, United States and all other chemicals and reagents used were of analytical grade.

Protein-A agarose, goat anti-rabbit IgG, 3, 3′, 5, 5′-tetramethylbenzidine (TMB), immobilon-P PVDF membrane, luminol, p-coumaric acid, Freund’s complete and incomplete adjuvants, testicular hyaluronidase, hyaluronic acid, o-dianisidine, horseradish peroxidase (HRP) 250U, and all chemicals were obtained from Sigma, St Louis, USA. Sodium chloride, bovine serum albumin, Folin-Ciocalteu reagent, Tris-HCl, casein, gelatin, trichloroacetic acid, sodium carbonate, calcium chloride, magnesium chloride, acrylamide and bis-acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate, tetramethyl ethylenediamine (TEMED), sodium formate, triton X-100, Alcian blue, acetic acid, triethanolamine, L-leucine, methanol, and all other chemicals were purchased from Sisco Research Laboratories (SRL), Mumbai, India. HRP-conjugated goat anti-horse IgG (H+L) was purchased from KINESISDx, South Paseo Dr, Brea, CA 90603, USA. Molecular weight markers were obtained from Genetix Biotech. Asia Pvt. Ltd. Bengaluru, India. LDH, CK, and CK-MB kits were purchased from AGAPPE Diagnostics Ltd. Kerala, India. Liquicelin-E and Uniplastin reagents were purchased from TULIP Diagnostics (P) Ltd. India.