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.
Np tosyl l phenylalanine chloromethyl ketone tpck
Manufactured by Merck Group
Sourced in United States
Np-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) is a laboratory reagent used in biochemical research. It is a serine protease inhibitor that selectively inhibits chymotrypsin-like proteases.
Automatically generated - may contain errors
Lab products found in correlation
Phenylmethylsulfonyl fluoride, by Merck Group (3 mentions)
Bapna, by Merck Group (1 mentions)
Nα tosyl l lysine chloromethyl ketone hydrochloride tlck, by Merck Group (1 mentions)
Sephadex g 50 fine, by Merck Group (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
50 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca protein estimation kit, by Thermo Fisher Scientific (1 mentions)
Snakeskin dialysis membrane, by Thermo Fisher Scientific (1 mentions)
Cnbr activated sepharose 4b, by Merck Group (1 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Casein, by Sisco Research Laboratories (1 mentions)
Gelatin, by Merck Group (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Polyvinyl pyrrolidone, by Sisco Research Laboratories (1 mentions)
Bovine serum albumin (bsa), by Sisco Research Laboratories (1 mentions)
α chymotrypsin, by Sisco Research Laboratories (1 mentions)
Amine coupling kit, by Cytiva (1 mentions)
Urea, by Cytiva (1 mentions)
6 protocols using np tosyl l phenylalanine chloromethyl ketone tpck
1
Purification and Characterization of Proteases
2
Cellular Signaling Pathways Regulation
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Phorbol myristate acetate (PMA) was obtained from Sigma–Aldrich (Sigma), dissolved in DMSO and stored at −20°C. The final concentration of dimethylsulphoxide (DMSO) in all experiments was less than 0.1%. The ERK inhibitor SB202190, NF-κB inhibitor, N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and p38 inhibitor SB203580 were purchased from Sigma–Aldrich. Caspase inhibitor Z-VAD-FMK was purchased from Selleck. Cell culture reagents and medium were obtained from Corning/Costar and BD-Falcon. The antibody against total ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), Caspase-1, Caspase-3, Caspase-9 (Human Specific) and ACTB were obtained from Cell Signaling Technology.
3
Purification and Characterization of PCB
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Essentially fatty acid free HSA (type A1887), and porcine trypsin (1800 U/mg) were purchased from Sigma-Aldrich (USA) and used without further purification. HSA concentration was determined using an extinction coefficient of 35 700 M-1 cm-1 at 280 nm. Protease inhibitors N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK; ≥97.0%) and phenylmethanesulfonyl fluoride (PMSF; ≥99.0%) were from Sigma-Aldrich. PCB was purified from commercial Hawaiian Spirulina pacifica powder (Nutrex, USA) and quantified by measuring absorbance at 680 nm as previously described [13 ]. All measurements were done in 20 mM Tris buffer, pH 7.4 (except for trypsin digestion study, see below). Final concentrations of methanol in HSA-PCB mixtures did not exceed 1% (v/v). All other chemicals were of analytical reagent grade and Milli-Q water (Millipore, Molsheim, France) was used throughout the experiments.
4
Glutathione-Mediated Detoxification Assay
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1-chloro-2, 4-dinitrobenzene (CDNB), phenylmethylsulfonylfluoride (PMSF), reduced glutathione (GSH), glutathione sepharose 4 fast flow, sephacryl S-300, cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, p-chloromercuribenzoic acid (pCMB), N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, gel filtration molecular weight markers, and triphenyltin chloride were purchased from Sigma Chemical Co. All other chemicals were of analytical grade.
5
MDCK Cell Line Cultivation for Viral Infection
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MDCK cells were purchased from the Korean Cell Line Bank (Korea; KCBL10034). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Welgene) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (100 U/ml; Sigma-Aldrich, USA) at 37°C and 5% CO2. For viral infection, the cells were cultured in DMEM supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, and trypsin treated with 2 μg/ml Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK; Sigma-Aldrich). Stock solutions of AS1842856 (Calbiochem, USA) were prepared in dimethyl sulfoxide (Sigma-Aldrich) and stored at −20°C.
6
Protocol for Cell Culture Reagents
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (HS), penicillin, streptomycin, G418, l -glutamine, dimethyl sulfoxide (DMSO), N-acetylcysteine (NAC), N-ω-nitro-l -arginine (NNLA), polyethylenoimine (PEI), collagen, pluronic, probenecid, Hank’s balanced salt solution (HBSS), Hepes, ethanol, phosphate-buffered saline (PBS), Thioflavin-T, sodium nitroprusside (SNP), D184 (Deano), TRI reagent, deoxyribonuclease I (DNase I), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride hydrate, bis Benzimide (Hoechst 33258), 3-(4,5-dimethyl-2-tiazolilo)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Tris-buffered saline, NaCl, Tween 20, 2,2′,2″,2″′-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), sodium fluoride (NaF), sodium orthovanadate (Na3VO4), sodium pyrophosphate (NaP2O3), benzamidine, Nonidet P-40, phenylmethylsulfonyl fluoride (PMSF), N-p-Tosyl-l -phenylalanine chloromethyl ketone (TPCK), soybean trypsin inhibitor (STI), aprotinin, leupeptin, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell Lysis Buffer (10×) was obtained from Cell Signaling (Beverly, MA, USA).
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