To test for potential degradation of IL-6, 10 µl conditioned culture medium obtained from infection experiments was used either untreated, heat-treated (95°C/5 min), or mixed with 8 µl of different protease inhibitors (or respective controls) and then incubated with 2 µl (0.5 µg/µl) recombinant IL-6 (Peprotech, Rocky Hill) at 37°C. For inhibition of metalloproteases, phosphoramidon (Sigma-Aldrich) was used at a final concentration of 3.4 mM. Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, Sigma-Aldrich), primarily inhibiting serine proteases, was used at a final concentration of 40 mM. Additionally, a proprietary, broad non-metalloprotease inhibitor was used (cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, Roche, one tab dissolved in 500 µl water).
Nα tosyl l lysine chloromethyl ketone hydrochloride tlck
Manufactured by Merck Group
Sourced in United States
Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) is a laboratory reagent commonly used as a serine protease inhibitor. It functions by irreversibly inhibiting the enzymatic activity of serine proteases.
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Lab products found in correlation
Phosphoramidon, by Merck Group (1 mentions)
Recombinant il 6, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Bapna, 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)
Np tosyl l phenylalanine chloromethyl ketone tpck, 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)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Polyvinyl pyrrolidone, by Sisco Research Laboratories (1 mentions)
Bovine serum albumin (bsa), by Sisco Research Laboratories (1 mentions)
5 protocols using nα tosyl l lysine chloromethyl ketone hydrochloride tlck
1
Evaluating IL-6 Stability with Protease Inhibitors
2
Purification and Characterization of Proteases
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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.
3
Affinity Purification of PorL-Myc Protein
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P. gingivalis porL/porL’-‘myc were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl. 0.5 M sucrose, 5 mM MgCl2, 1% DDM, 1x complete protease inhibitor cocktail (Roche), benzonase, 250 units (Sigma-Aldrich) and 5 mM Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK, Sigma-Aldrich) and incubated on ice for 45 min. Unlysed cells and debris were removed by centrifugation at 10000 g for 25 min at 4°C. The lysate was then incubated with Myc agarose (MBL International) for 1 hour at 4°C with rotation. The agarose was then treated with 0.5 mg/ml lysozyme in 50 mM Tris pH 7.5, 500 mM NaCl, 1% DDM, 5 mM EDTA, and incubated at 37°C for 15 min followed by washes in 50 mM Tris-HCl, 150 mM NaCl and 0.1% DDM. The agarose was then either resuspended in SDS loading buffer and heated at 95°C for 10 min or was incubated with 40 μL of myc peptide (1 mg/mL for 10 min on ice to elute the complexes bound to the Myc agarose). The eluted complex was then separated by SDS-PAGE and the bands were identified by mass spectrometry.
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P. gingivalis porL/porL’-‘myc were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl. 0.5 M sucrose, 5 mM MgCl2, 1% DDM, 1x complete protease inhibitor cocktail (Roche), benzonase, 250 units (Sigma-Aldrich) and 5 mM Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK, Sigma-Aldrich) and incubated on ice for 45 min. Unlysed cells and debris were removed by centrifugation at 10000 g for 25 min at 4°C. The lysate was then incubated with Myc agarose (MBL International) for 1 hour at 4°C with rotation. The agarose was then treated with 0.5 mg/ml lysozyme in 50 mM Tris pH 7.5, 500 mM NaCl, 1% DDM, 5 mM EDTA, and incubated at 37°C for 15 min followed by washes in 50 mM Tris-HCl, 150 mM NaCl and 0.1% DDM. The agarose was then either resuspended in SDS loading buffer and heated at 95°C for 10 min or was incubated with 40 μL of myc peptide (1 mg/mL for 10 min on ice to elute the complexes bound to the Myc agarose). The eluted complex was then separated by SDS-PAGE and the bands were identified by mass spectrometry.
4
RIG-I Trypsin Proteolysis Assay
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To carry out trypsin proteolysis, 400 ng of purified RIG-I with or without cFP (1 mM) was mixed with 1 μg of HCV 3′-UTR RNA or poly(I:C) in a 20-μl reaction buffer (20 mM Tris–HCl, pH 8.0, 1.5 mM MgCl2, 1.5 mM DTT, and 70 mM KCl) supplemented with 6.7 µg of AMP-PNP (Sigma). After 5 min incubation at room temperature, sequencing grade TPCK-treated modified trypsin (Promega) was added to the reaction mixture at a RIG-I:trypsin ratio of 100:1 (w/w) and further incubated at 37 °C. The reaction was terminated by addition of 0.5 μg of N-α-Tosyl-L -lysine chloromethyl ketone hydrochloride (TLCK) (Sigma), which is an irreversible inhibitor of trypsin. After 5 min incubation at room temperature, protein samples were subjected to SDS-14% PAGE followed by immunoblotting analysis using a RIG-I-specific antibody.
5
Tumor Dissociation and Spheroid Culture
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Freshly resected tumor samples were diced using a razor blade and incubated for 30 min at 37°C in a tissue culture dish (100-mm diameter) with digestion buffer, consisting of 4 ml of HBSS (Gibco) supplemented with 8 mg of collagenase D (Sigma-Aldrich), 80 µg DNase I (Sigma-Aldrich), and 40 µg Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK;Sigma-Aldrich) per ∼2 g of the tumor sample. The sample was mixed by pipetting up and down several times every 10 min. Then, the cell suspension was mechanically dissociated using a tissue homogenizer (Potter-Elvehjem polytetrafluoroethylene pestle) in HBSS. Cells were cultured ex vivo as tumor spheroids in Neurobasal Medium with 1% B27 supplement, 0.25% N2 supplement, 1% penicillin streptomycin, 1 µg/ml heparin, 20 ng/ml human basic fibroblast growth factor, and 20 ng/ml human epidermal growth factor. Peripheral blood was processed by density gradient separation (Ficoll) to obtain PBMCs.
For tumor lysate preparation, tumor single-cell suspension was resuspended at 106 cells/ml of PBS and underwent five freeze-thaw cycles and 1 min of sonication.
For tumor lysate preparation, tumor single-cell suspension was resuspended at 106 cells/ml of PBS and underwent five freeze-thaw cycles and 1 min of sonication.
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