Takara bradford protein assay kit

The activities of β-lactamase were determined by spectrophotometer using nitrocefin (Nitrocefin; Kanto Chemical, Tokyo, Japan) [18 (link)]. A 10-μl aliquot of supernatant (crude enzyme solution) was added 990 μl of 100 μM nitrocefin solution, and the delta optical density (O.D.) of OD 486 nm was measured. The specific activities of β-lactamase were expressed in micromoles of nitrocefin hydrolyzed per minute per milligram of protein based on the changes in the absorbance at 486 nm at 35 °C for 3 min (ΔOD/sec). The supernatant protein concentrations were determined using the Bradford protein assay kit (TaKaRa Bradford Protein Assay Kit; TAKARA Bio Inc., Shiga, Japan). Bovine serum albumin (Bovine Serum Albumin; TAKARA Bio Inc., Shiga, Japan) was used as the standard. The ratio of the specific activities of β-lactamase and those of controls without antibiotics were used. The E. coli Mec5372 strain and C. koseri Rck4438 strain have co-production of TEM-1, and K. pneumoniae Rkp2004 strain have co-production of TEM-1 and SHV-12, so the β-lactamase activities of these strains were determined by a spectrophotometer using the nitrocefin solution after adding 4 μg/ml clavulanic acid (CVA; GlaxoSmithKline K.K., Tokyo, Japan).

Cells cultured in fruit juice supplemented with arginine for 48 h were harvested and suspended into a 50 mM phosphate buffer (pH 7.5). The cell suspension was sonicated, and cell-free extracts were obtained. The protein concentrations of the cell-free extracts were measured using the TaKaRa Bradford Protein Assay Kit (Takara Bio Co., Ltd., Shiga, Japan). Arginine deiminase activity was assayed by measuring the rate of arginine change during the incubation for 40 min at 30 °C in a reaction mixture containing a 100 mM citrate buffer (pH 5.5) with 5 mM MnCl₂, 5 mM L-arginine, and the cell extracts. Ornithine carbamoyl-transferase activity was assayed by measuring the conversion rate of ornithine changed during the incubation for 40 min at 30 °C of reaction mixtures containing a 150 mM imidazole buffer (pH 7.5) with 10 mM carbamoyl phosphate, 10 mM L-ornithine, 10 mM phosphate, and cell extracts. The concentration of arginine changed to ornithine was measured via HPLC.

Total cell lysates were extracted using lysis buffer [Tris-HCl (pH 7.4), sodium dodecyl sulfate (SDS), mercaptoethanol, and glycerol]. Protein concentrations were determined using Takara Bradford Protein Assay kit (T9310A; Takara Bio, Shiga, Japan), and an equal amount of protein (10 µg) was separated on a 4–20% gradient SDS-PAGE (TEFCO, Tokyo, Japan). Proteins were transferred using a Trans-Blot Turbo RTA Mini LF PVDF Transfer kit (170-4274; Bio-Rad Laboratories, Hercules, CA, USA) to a Trans-Blot Turbo Mini-size LF polyvinylidene difluoride membrane (Bio-Rad Laboratories) and blocked with 5% non-fat dry milk in Tris-buffered saline [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween-20] for 1 h at room temperature. Membranes were then incubated with anti-KHSRP and anti-myc primary antibodies at dilutions of 1:1,000 in 5% non-fat dry milk in Tris-buffered saline overnight at 4°C. Following incubation with appropriate secondary antibodies conjugated with horseradish peroxidase (sc-2004, sc-2005; Santa Cruz Biotechnology) at dilutions of 1:2,000 for 1 h at room temperature, immunoreactive bands were visualized using the ECL Plus kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions.

Eighteen fish specimens from three representative ciguateric fish species (L. monostigma, L. bohar, and O. punctatus) and two non-ciguateric fish species (L. gibbus and L. fulviflamma) were collected from the Ryukyu Islands in Okinawa Prefecture. The liver was removed from each fish and promptly used for S9 fraction preparation. Individual liver samples were weighed and homogenized in two times the volume of ice-cold buffer (20 mM Tris, 0.15 M KCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM benzamidine, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 10% glycerol) using a glass-Teflon homogenizer. The homogenate was then centrifuged at 9000× g for 10 min at 4 °C and the resultant supernatant (S9 fraction) was ultracentrifuged at 100,000× g for 60 min at 4 °C. The microsome pellet was resuspended and homogenized in ice-cold buffer. The protein was assayed with a commercially available kit (TaKaRa Bradford Protein Assay Kit; TaKaRa, Japan) according to the manufacturer’s instructions, using bovine serum albumin as a standard. The S9 and microsomal fractions were then stored in small aliquots at −80 °C until required for use. All preparation steps of the enzyme sources were carried out on ice.