The TAMRA-fluorophosphonate activity-based probe was obtained from ThermoFisher. N-(trans-epoxysuccinyl)-l -leucine-4-guanidinobutylamide (E-64), AA74-1, clindamycin, chloroquine, and isopentenyl pyrophosphate trilithium salt were purchased from Sigma. Pepstatin A was purchased from MP Biomedicals.
N trans epoxysuccinyl l leucine 4 guanidinobutylamide e 64
Manufactured by Merck Group
Sourced in United States
N-(trans-epoxysuccinyl)-l-leucine-4-guanidinobutylamide (E-64) is a protease inhibitor that selectively and irreversibly inhibits cysteine proteases, including papain, cathepsins B, H, and L, and calpains. It is a useful research tool for studying the role of cysteine proteases in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (2 mentions)
Pepstatin a, by MP Biomedicals (1 mentions)
Clindamycin, by Merck Group (1 mentions)
Mono q, by GE Healthcare (1 mentions)
Calpeptin, by Merck Group (1 mentions)
N ethylmaleimide nem, by Merck Group (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Bestatin, by Merck Group (1 mentions)
Paromomycin, by Merck Group (1 mentions)
Ornidazole, by Merck Group (1 mentions)
Emetine, by Merck Group (1 mentions)
Dnazol reagent, by Thermo Fisher Scientific (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using n trans epoxysuccinyl l leucine 4 guanidinobutylamide e 64
1
TAMRA-fluorophosphonate Probe Activity
2
Recombinant Protein Purification and Activation
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InpA was produced as a recombinant protein in Escherichia coli and purified as previously described [12 (link), 13 (link)] by affinity chromatography on Fast Flow Ni-NTA (Ni2+-nitrilotriacetate)–Sepharose (Qiagen) followed by anion exchange chromatography (MonoQ, GE Healthcare). The active concentration of InpA was determined by active-site titration using an appropriate dilution of a standardized 1 mM aqueous solution of the protease inhibitor N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E-64) (Sigma-Aldrich; product E3132) needed for total inactivation of the proteinase. Residual enzyme activity was determined by measurement of fluorescence (λex = 380 nm and λem = 460 nm) of AMC (7-amino-4-methylcoumarin) released from t-butoxycarbonyl-Val-Leu-Lys-AMC as described previously [13 (link)]. Immediately before use, InpA was activated by incubation for 15 min with 3 mM DTT in 0.1 M Tris–HCl, 0.14 M NaCl, pH 7.5 [50 ]. When used for protein breakdown assays under non-reducing conditions, the above buffer was exchanged for that without reducing agent by ultrafiltration using 10 kDa cut-off Microcons (Amicon).
3
Protease Inhibitor Cocktail for In Vitro and In Vivo Studies
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4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlorine (AEBSF, A8456), aprotinin (A1153), bestatin (Sigma B8385), pepstatin A (P5318), N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64, E3132), leupeptin (L2884), N-ethylmaleimide (NEM, E3876), calpeptin (C8999), calpain inhibitor-I (A6185), -II (A6060), -III (M6690), pan-caspase inhibitor (V116), BAPTA-AM (A1076), ionomycin (I0634) were supplied by Sigma. Calpistatin peptide (208902), negative control peptide (208904), elastase inhibitor IV (324759) and cathepsin G inhibitor I (219372) were supplied by Calbiochem. Tryptase inhibitor nafamostat mesylate (NM, 3081) was supplied by Tocris. AEBSF, aprotinin, bestatin, pepstatin A, E64, leupeptin and NEM were used in vitro at concentrations according to manufacturer’s instructions (Sigma Inhib1). calpeptin, calpain, NE, CG, caspase and tryptase inhibitors, BAPTA-AM were used at 100 μM during in vitro studies. Calpistatin and control peptide were used at 10 μM during in vitro studies. Doses of inhibitors used in vivo and details of all other reagents used are described in other method sections.
4
Preparation and Storage of Anti-Parasitic Compounds
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MTZ, ornidazole, emetine, chloroquine, paromomycin, L-cysteine, and N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E-64) were purchased from Sigma-Aldrich (St. Louis, MO, USA), while tinidazole was acquired from LTK Laboratories (St. Paul, MN, USA). DNAzol reagent, TRIzol reagent, PLUS reagent, Lipofectamine, and geneticin (G418) were secured from Invitrogen (Carlsbad, CA, USA). All other chemicals were obtained from Wako Pure Chemical Industries (Osaka, Japan) unless otherwise stated. Drugs were dissolved either in distilled water or dimethyl sulfoxide (DMSO) to a stock concentration of 100 mM and stored at −30°C.
5
Papain Treatment of Semen Samples
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Fifteen minutes after the initial dilution and pipetting, the ejaculates were split into two 15 mL tubes. In one tube (Pap), Papain from Carica Papaya (Sigma-Aldrich, St. Louis, MO, USA) was added (final concentration of 0.1 mg/mL [22 (link)]), whereas the other tube was kept as a control (Con), TCF was added with the same volume of added Papain. Both the control and Papain-treated samples were pipetted, with the same number of pipetting repetitions (no. = 10) soon after dilution, and twice more (no. = 10 pipetting) in the following 5 min. After 5 min, the Papain reaction was stopped by adding N-(trans-Epoxysuccinyl)-l -leucine 4-guanidinobutylamide (E-64; Sigma-Aldrich, St Louis, MO, USA) (final concentration 10 µM) and TCF of the same volume of the E64 solution was added to the Con sample [22 (link)].
When observed, the sperm head-to-head agglutination was scored from 0 to 5 according to the following criteria: 0-absence of agglutination; 0.5 (5% agglutinated sperm); 1 (10% agglutinated sperm); 1.5 (15%); 2 (20%); 2.5 (25%) 3 (30%) and 4 (40%); 5 (≥50%) (modified from [20 (link)]).
The two samples (Con, Pap) were again evaluated for viability and concentration. Two hundred microliter aliquots were again collected from each tube, diluted with TCF-3% BSA, incubated for 5 min at 37 °C, and evaluated for motility and kinematic parameters as described before.
When observed, the sperm head-to-head agglutination was scored from 0 to 5 according to the following criteria: 0-absence of agglutination; 0.5 (5% agglutinated sperm); 1 (10% agglutinated sperm); 1.5 (15%); 2 (20%); 2.5 (25%) 3 (30%) and 4 (40%); 5 (≥50%) (modified from [20 (link)]).
The two samples (Con, Pap) were again evaluated for viability and concentration. Two hundred microliter aliquots were again collected from each tube, diluted with TCF-3% BSA, incubated for 5 min at 37 °C, and evaluated for motility and kinematic parameters as described before.
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