Dulbecco's minimum essential medium (DMEM), glutamine and penicillin/streptomycin/glutamine stock mix were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and charcoal-stripped FBS were from Invitrogen (Carlsbad, CA, USA). L-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) was from Sigma-Aldrich (St. Louis, MO USA). CTSO, MTDH, PABPC4L, LMNA, EEFiA1and control small interfering RNAs (siRNA) were purchased from Dharmacon (Thermo Scientific Dharmacon, Inc.). CTSO plasmid was purchased from OriGene (Rockville, MD, USA). Affinity purified rabbit and mouse antibodies against human BRCA1 and CTSO were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). ZNF423 antibody was purchased from Abcam (Cambridge, MA, USA). Actin, MTDH, PABPC4L, LMNA, and EEFiA1 antibodies were from cell signaling (Danvers, MA, USA). For standard PCR, HotStart Taq Plus DNA Polymerase was used (Qiagen, Germantown, MD, USA). Reagents and primers for real time PCR were purchased from Qiagen (Valencia, CA, USA). The protease inhibitor cocktail kit was obtained from Pierce Biotechnology (Rockford, IL, USA). 17β-estradiol (E2) and 4-hydroxytamoxifen (OH-TAM) were purchased from Sigma Aldrich (Saint Louis, MO USA). Olaparib was from Selleckchem (Houston, TX, USA).
L trans epoxysuccinyl leucylamido 4 guanidino butane e 64
Manufactured by Merck Group
Sourced in United States
L-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) is a lab equipment product. It is a protease inhibitor that specifically and irreversibly inhibits cysteine proteases.
Automatically generated - may contain errors
Lab products found in correlation
Ca 074, by Merck Group (2 mentions)
Z phe arg amc, by Merck Group (2 mentions)
Sly amc, by Cayman Chemical (2 mentions)
N succinyl leu tyr 7 amido 4 methylcoumarin, by Cayman Chemical (2 mentions)
Ac lly afc, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Actin, by Cell Signaling Technology (1 mentions)
4 hydroxy tamoxifen oh tam, by Merck Group (1 mentions)
Charcoal stripped fbs, by Thermo Fisher Scientific (1 mentions)
Ctso plasmid, by OriGene (1 mentions)
Hotstarttaq plus dna polymerase, by Qiagen (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
17β estradiol e2, by Merck Group (1 mentions)
A spectrometer, by Agilent Technologies (1 mentions)
Spectrometer, by Agilent Technologies (1 mentions)
Glycine, by Merck Group (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Bovine serum albumin bsa, by ICN Biomedicals (1 mentions)
β glucosidase, by Merck Group (1 mentions)
6 protocols using l trans epoxysuccinyl leucylamido 4 guanidino butane e 64
1
Gene Expression Regulation in Breast Cancer
2
Measuring CTSL Activity with Fluorometric Assay
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Cells were suspended in ice-cold lysis buffer (400 mM sodium phosphate buffer, pH 6.0, 75 mM NaCl, 4 mM EDTA, and 0.25% Triton X-100) and incubated on ice for 30 min. Lysates were centrifuged at 15,000 rpm for 20 min at 4 °C. Protein concentration was determined by the BCA protein assay kit (Pierce, Rockford, IL). CTSL activity was measured by fluorometric assay [35 (link)]. Briefly, 10 μg of total cell lysate was diluted in 100 μL of 0.34 M sodium acetate buffer, pH 5.5, containing 2 mM EDTA, and 4 mM dithiothreitol (DTT). To discriminate between CTSL and cathepsin-B activities, a selective cathepsin-B inhibitor, CA074 (Sigma), was added at a final concentration of 5 μM and pre-incubated for 15 min at 37 °C. Fluorogenic substrate Z-Phe-Arg-AMC (Sigma) was added to a final concentration of 5 μM and samples were incubated for an additional 30–60 min at 37 °C. Fluorescence of the degradation product, 7-amino-4-methylcoumarin (7-AMC), was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm, using a spectrometer (BioTeK). Cysteine protease inhibitor L-trans-epoxy-succinyl-leucylamido-(4-guanidino)-butane (E64, Sigma) was used as a CTSL inhibitor at 50 μM concentration.
3
Fluorometric Assay for CTSL Activity
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Cells were suspended in ice-cold lysis buffer (400 mM sodium phosphate buffer, pH 6.0, 75 mM NaCl, 4 mM EDTA, and 0.25 % Triton X-100) and incubated on ice for 30 minutes. Lysates were centrifuged at 15,000 rpm for 20 minutes at 4 °C. Protein concentration was determined by the BCA protein assay kit (Pierce, Rockford, IL). CTSL activity was measured by uorometric assay [30] . Brie y, 10 µg of total cell lysate was diluted in 100 µL of 0.34 M sodium acetate buffer, pH 5.5, containing 2 mM EDTA, and 4 mM dithiothreitol (DTT). To discriminate between CTSL and cathepsin-B activities, a selective cathepsin-B inhibitor, CA074 (Sigma), was added at a nal concentration of 5 µM and pre-incubated for 15 minutes at 37 °C. Fluorogenic substrate Z-Phe-Arg-AMC (Sigma) was added to a nal concentration of 5 µM and samples were incubated for an additional 30-60 minutes at 37 °C. Fluorescence of the degradation product, 7-amino-4-methylcoumarin (7-AMC), was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm, using a spectrometer (BioTeK). Cysteine protease inhibitor l-transepoxy-succinyl-leucylamido-(4-guanidino)-butane (E64, Sigma) was used as a CTSL inhibitor at 50 µM concentration.
4
Calpain Activity Assay with Fluorogenic Substrates
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Calpain proteolytic activity was measured by hydrolysis of the synthetic calpain substrates, sLY-AMC (N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin; Cayman) and Ac-LLY-AFC (Ac-Leu-Leu-Tyr-7-amino-4-trifluoromethylcoumarin; Fisher) (Wert et al., 2015 (link)). Briefly, 10 μM or 0.25 μM of purified calpain was added to a reaction buffer containing 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 2 mM DTT, and 0.1 to 100 mM CaCl2 or 5 mM EDTA and incubated on ice prior to reaction in 96-well plates (100 μL per reaction). Varying concentrations of substrate (10 μM to 1 mM) were added and the reaction was incubated at 37°C for 1 hour on a fluorimetric plate reader (Tecan Spark, Männedorf Switzerland). All experiments were performed in triplicate. The amount of AMC or AFC released during the experiment was calculated based on a standard curve of AMC or AFC concentrations (from 2 nM to 5 μM) and kinetic parameters were calculated by direct fitting to the Michaelis-Menten equation in GraphPad Prism 8. Inhibition experiments were carried out in similar manner with either E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (Sigma) or the human CAST B27 domain (residues 184–210; Calbiochem) in the presence of 0.2 mM Ac-LLY-AFC. Reaction rates at various inhibitor concentrations (10 nM to 10 μM) were normalized and fit to the Hill equation in GraphPad Prism 8.
5
Calpain Activity Assay with Fluorogenic Substrates
6
Fluorogenic Substrate Synthesis and Assay
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4-Methylumbelliferone sodium salt (4-MU), 7-amino-4-methylcoumarin (4-MC), 4-MU-β-D-galactoside, dimethyl sulfoxide, Triton X-100, DL-dithiothreitol, sodium azide, β-glucosidase, pepstatin A, E64 (trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane), and ethylenediamine were purchased from Sigma-Aldrich Corp. (St. Louis, MO. USA). 4-MU-6-thio-palmitate-β-D-glucopyranoside was purchased from Carbosynth (United Kingdom). Ala-Ala-Phe-7-amido-4-methylcoumarin was purchased from Bachem (United Kingdom). Sodium carbonate, glycine, sodium hydroxide, acetic acid, sodium acetate, citric acid, sodium phosphate, sodium chloride, chloroform, and methanol were from Merck (Darmstadt, Germany). Bovine serum albumin (BSA) was purchased from ICN Biomedicals (Aurora, OH, USA). ethylenediaminetetraacetic acid (EDTA) was purchased from Panreac (Barcelona).
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