Enzchek protease assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The EnzChek Protease Assay Kit is a laboratory product designed to measure protease activity. It provides a sensitive fluorometric method for detecting and quantifying protease activity in a variety of sample types.

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Lab products found in correlation

Dispase, by Roche (3 mentions) Varioskan fluorescent plate reader, by Thermo Fisher Scientific (2 mentions) Novex zymogram casein gels, by Thermo Fisher Scientific (2 mentions) Spectromax gemini xs, by Molecular Devices (1 mentions) Softmax pro software, by Molecular Devices (1 mentions) Microplate reader, by Tecan (1 mentions) Pancreatin from porcine pancreas, by Merck Group (1 mentions) Pepsin from porcine gastric mucosa, by Merck Group (1 mentions) Papain, by Merck Group (1 mentions) Spectramax gemini xs, by Molecular Devices (1 mentions) Streptavidin conjugated horseradish peroxidase, by R&D Systems (1 mentions) 96 well plate, by Corning (1 mentions) Glomax plate reader, by Promega (1 mentions) 3 3 5 5 tetramethylbenzidine substrate, by BD (1 mentions) Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions) Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions) Er tracker blue white dpx, by Thermo Fisher Scientific (1 mentions) Live dead fixable near ir stain, by Thermo Fisher Scientific (1 mentions) Infinite m nano plate reader, by Tecan (1 mentions) Caspase glo 3 7 assay, by Promega (1 mentions)

32 protocols using enzchek protease assay kit

Fluorescent Protease Activity Assay

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The intestinal homogenates, along with purified proteases, were tested for general proteolytic activities (metallo-, serine, acid and sulfhydryl proteases) using casein derivative substrate (Enzchek Protease Assay Kit, E6639, Molecular Probes-Invitrogen, Carlsbad, CA).
The substrate in the Enzchek Protease Assay Kit, casein, is derivatized with pH-insensitive fluorophores. When undigested, the fluorophores are quenched and the molecule does not fluoresce. Upon cleavage by proteases, the peptides with these fluorophores will fluoresce. The level of measured fluorescence increases with the amount of peptide present. The fluorescence of the intestinal homogenates incubated with this substrate was measured in duplicates on a 96-well plate by a spectrophotometer (Spectromax Gemini XS) using the Softmax Pro software (Molecular Devices Corp., Sunnvale, CA) and expressed in relative fluorescent units (RFUs). The level of fluorescence increases with the number of sites within the casein molecule cleaved by the enzymes in the sample.
In each well of a multiwell plate, 5 μl of intestinal sample, 95 μl of digestion buffer and 100 μl of casein substrate were mixed. The plate was covered with aluminum foil to prevent evaporation, incubated at 37°C in a spectrophotometer (protected from light), and kinetic measurements were made over a period of 60 minutes.

Chan A, & Schmid-Schönbein G.W. (2019). Pancreatic Source of Protease Activity in the Spontaneously Hypertensive Rat and its Reduction During Temporary Food Restriction. Microcirculation (New York, N.Y. : 1994), 26(6), e12548.

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Quantitative Extracellular Protease Assay

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The quantitative extracellular protease activity in the solution was investigated in Renuspore^® by employing a commercial EnzChek^® Protease Assay Kit (Molecular Probes, Eugene, OR, USA), which contains a casein derivative labeled with green-fluorescent BODIPY^® FL dye.
The cells of Renuspore^® and L. rhamnosus GG (1 x 10⁸ CFU/mL) were washed and suspended in 100 μl of 1X PBS for the assay. Proteinase K was used as a positive control, and all samples were assayed with an incubation time of 24 h using the EnzChek^® Protease Assay Kit, according to the manufacturer's instructions. Fluorescence (Ex/ Em 485/ 535 nm) was measured on a Tecan Microplate Reader using standard filters.

Simon A., Colom J., Mazhar S., Khokhlova E., Deaton J, & Rea K. (2023). Bacillus megaterium Renuspore® as a potential probiotic for gut health and detoxification of unwanted dietary contaminants. Frontiers in Microbiology, 14, 1125616.

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Protease Activity Assay with Casein

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Protease activity against casein was tested using the EnzChek protease assay kit (Molecular probes #E6638, green fluorescent casein substrate). The working solution was prepared by diluting the stock to 10 µg/ml in 50 mM sodium citrate, pH 4.5. The purified protease fractions were diluted with sodium citrate buffer. 100 µl of the diluted substrate was combined with the diluted protease fractions in a 96 well sample plate. The plate was then covered and kept at 37 °C for 1–3 h. Fluorescence readings were taken at 1, 2, and 3 h with a Varioskan fluorescent plate reader (Thermo Scientific) using 485 nm excitation and 530 nm emission.

Landowski C.P., Mustalahti E., Wahl R., Croute L., Sivasiddarthan D., Westerholm-Parvinen A., Sommer B., Ostermeier C., Helk B., Saarinen J, & Saloheimo M. (2016). Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei. Microbial Cell Factories, 15, 104.

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Gastric Protease Activity Quantification

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Gastric aspirate samples were thawed on ice, centrifuged (3000 rpm; 4 °C) and the cream layer was removed⁵³. pH of the supernatant was determined (n = 425 samples) and samples were centrifuged to remove any remaining cream fraction (14,000 rpm; 4°C)⁵³. Total protease (n = 433 samples) and pepsin activity (n = 434 samples) were determined using the green fluorescence EnzChek Protease Assay Kit (Molecular Probes, Eugene, OR, USA) in duplicate according to manufacturer’s instructions. For determining total protease activity, 10mM TRIS buffer (pH 7.8) was used and the standard curve was generated using pancreatin from porcine pancreas (Sigma-Aldrich)⁵³. For determining pepsin activity, 10mM HCl buffer with pH 2.2 was used and the standard curve was generated using pepsin from porcine gastric mucosa (Sigma-Aldrich)⁵³.

Henderickx J.G., Zwittink R.D., Renes I.B., van Lingen R.A., van Zoeren-Grobben D., Jebbink L.J., Boeren S., van Elburg R.M., Knol J, & Belzer C. (2021). Maturation of the preterm gastrointestinal tract can be defined by host and microbial markers for digestion and barrier defense. Scientific Reports, 11, 12808.

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Quantifying Protease Activity Using EnzChek Assay

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The EnzChek protease assay kit (Molecular Probes) was used to measure protease activity. Tag-free SDE1, E-64 (Sigma-Aldrich), and BSA (Gold Biotechnologies) at two different concentrations (100 and 500 nM) were mixed with papain (Sigma-Aldrich) at 100 μg/mL and added to 96-well Immulon plates (Thermo Scientific) containing BODIPY FL casein substrate. papain with MES buffer alone served as a no treatment control for proteolytic activity and SDE2 (CLIBASIA_03230) at 300 nM served as an alternative CLas effector control. Reactions were allowed to perform for 1 h at room temperature in the dark before fluorescence was measured using a Tecan Pro 2000 plate reader at 460/480 nm excitation/emission, with a gain value of 50. p-values were determined using a two-tailed Student’s t-test. SDE1 and SDE2 recombinant proteins were purified from E. coli using His60 Ni-NTA Superflow resins (Clontech). The purified SDE1 proteins were cleaved with Ubiquitin-like-specific protease 1 to remove the His-SUMO tag, generating tag-free SDE1 proteins.
The experiments were repeated at least three times with similar results. Uncropped raw data are presented in Supplementary Fig. 11.

Clark K., Franco J.Y., Schwizer S., Pang Z., Hawara E., Liebrand T.W., Pagliaccia D., Zeng L., Gurung F.B., Wang P., Shi J., Wang Y., Ancona V., van der Hoorn R.A., Wang N., Coaker G, & Ma W. (2018). An effector from the Huanglongbing-associated pathogen targets citrus proteases. Nature Communications, 9, 1718.

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Plasma Protease Activity Assay

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To assess the ability of plasma proteases to digest large globular proteins, we utilized the Enzchek protease assay kit (Enzchek BODIPY, casein derivative, catalogue no. E-6638; Molecular Probes, Carlsbad, CA, USA; cleaved by metallo-, serine, acid, and sulfhydryl proteases), which consists of casein internally quenched with Texas FITC fluorophores (Ex/Em: 505/513 nm) reconstituted in a digestion buffer. Plasma samples were tested simultaneously for overall protease activity. Protease activity levels were determined from fluorescent intensity after peptide cleavage following 18 h incubation at 37 °C (SpectraMax Gemini XS; Molecular Devices, Sunnyvale, CA, USA).

Kuo C.S., Chen J.S., Lin L.Y., Schmid-Schönbein G.W., Chien S., Huang P.H., Chen J.W, & Lin S.J. (2020). Inhibition of Serine Protease Activity Protects Against High Fat Diet-Induced Inflammation and Insulin Resistance. Scientific Reports, 10, 1725.

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Protease Activity Quantification Assays

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Protease activity against casein was tested using the EnzChek protease assay kit (Molecular probes #E6638, green fluorescent casein substrate) or the QuantiCleave protease assay kit (Pierce #23263, succinylated casein). The working stock solution was prepared by diluting the stock to 10 μg/ml in 50 mM sodium citrate, pH 4.5 or pH 5.5. The purified protease fractions were diluted with sodium citrate buffer. 100 μl of the diluted substrate was combined with the diluted protease fractions in a 96 well sample plate. The plate was then covered and kept at 37°C for one to three hours. Fluorescence readings were taken at one, two, and three hours with a Varioskan fluorescent plate reader (Thermo Scientific) using 485 nm excitation and 530 nm emission. To measure the QuantiCleave assay reaction 50 μl of TNBSA reagent was added to every well and the plate was incubated at 37°C. The absorbance at 450 nm was measured for the whole plate. Control wells with supernatant without substrate were used as background controls. The nonspecific background signal was subtracted from specific protease activity measurement.

Landowski C.P., Huuskonen A., Wahl R., Westerholm-Parvinen A., Kanerva A., Hänninen A.L., Salovuori N., Penttilä M., Natunen J., Ostermeier C., Helk B., Saarinen J, & Saloheimo M. (2015). Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei. PLoS ONE, 10(8), e0134723.

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Measuring Antigen-Processing in IgM+ B Cells

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The antigen-processing capacity of IgM⁺ B cells was measured using the EnzChek Protease Assay Kit (Invitrogen, Carlsbad, California, USA), as reported in previous studies (33 (link), 43 (link), 44 (link)). To summarize, AK leukocytes from tilapia at a concentration of 2 × 10⁶ cells/ml, obtained as described above, were incubated with green fluorescent BODIPY DQ-Casein™ at 5 μg/ml for 1 h. The cells were then washed three times with FACS staining buffer (Miltenyi Biotec; GmbH, Germany) and incubated with AF647-anti-IgM mAb (0.25 μg/ml) for 30 min at 4°C (35 (link)). After being washed three times, the cells were analyzed by flow cytometry as described above.

Wu L., Yang Y., Gao A., Li J, & Ye J. (2022). Teleost fish IgM+ plasma-like cells possess IgM-secreting, phagocytic, and antigen-presenting capacities. Frontiers in Immunology, 13, 1016974.

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Quantitative Analysis of Kallikrein Proteases

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For these experiments, we used primary keratinocytes cultures grown in low and high calcium conditions (see above). In KLK Elisa assays, 96-well plates (Corning, Corning, NY) were coated with rabbit anti-KLK5 or KLK7 antibody (Santa Cruz Biotechnology) at 1 μg/ml in phosphate-buffered saline (PBS). The wells were washed, blocked with PBS containing 1% BSA, and then incubated for 3 h with media isolated from keratinocytes grown for 12 hours in fresh media. Biotinylated goat anti-KLK5 or anti-KLK7 antibodies (R&D Systems) were used as detection antibodies. Colorimetric quantification at 450 nm was carried out by incubating plates with streptavidin-conjugated horseradish peroxidase (R&D Systems, Minneapolis, MN) and 3,3′5,5′-tetramethylbenzidine substrate (BD Biosciences, San Jose, CA). The reactions were stopped by 0.2 M sulfuric acid (Sigma-Aldrich, St. Louis, MO). The enzymatic activity of KLK proteins was measured by an EnzChek Protease Assay Kit (green fluorescence, Invitrogen) following the manufacturer’s recommendations. Absorbance or fluorescence was measured on a Glomax plate reader (Promega, Madison, WI)

Zhang L., Ferreyros M., Feng W., Hupe M., Crumrine D.A., Chen J., Elias P.M., Holleran W.M., Niswander L., Hohl D., Williams T., Torchia E.C, & Roop D.R. (2016). Defects in Stratum Corneum Desquamation Are the Predominant Effect of Impaired ABCA12 Function in a Novel Mouse Model of Harlequin Ichthyosis. PLoS ONE, 11(8), e0161465.

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Protease Activity of Sap2 Variants

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Complement activation was carried out as previously reported.^[⁹, ¹⁰^] Briefly, to study the effect of Val273Leu variation of Sap2 on complement activation, NHS was first pre‐treated with an equal amount of Sap2 secreted by different clinical strains, Sap2‐273V or Sap123 ko strains for 15 min at 37 °C, then added to 96‐well plates pre‐coated with lipopolysaccharide (LPS, 10 µg mL⁻¹) and further incubated for 60 min at 37 °C. The MAC formation was then quantified by polyclonal rabbit anti‐MAC antibody (1:3000) together with HRP‐labeled goat anti‐rabbit as a secondary antibody (1:2000). PBS used in the assay indicated a negative background control without NHS in the sample.
Protease activity of Sap2_273V and Sap2_273L at pH 5 and pH 7 was detected using a Protease Assay Kit (Invitrogen EnzChek Protease Assay Kit). Specifically, fluorescence‐quenched BODIPY FL casein (10 µg mL⁻¹) was first incubated with 1 µg recombinant Sap2_273V and Sap2_273L in PBS (pH 5 or 7) for 60 min at 25 °C, then the fluorescence was measured in a fluorescence microplate reader, using excitation and emission filters of 485 ± 12.5 and 530 ± 15 nm, respectively.

Lin L., Wang M., Zeng J., Mao Y., Qin R., Deng J., Ouyang X., Hou X., Sun C., Wang Y., Cai Y., Li M., Tian C., Zhou X., Zhang M., Fan H., Mei H., Sarapultsev A., Wang H., Zhang G., Zipfel P.F., Hu Y., Hu D, & Luo S. (2023). Sequence Variation of Candida albicans Sap2 Enhances Fungal Pathogenicity via Complement Evasion and Macrophage M2‐Like Phenotype Induction. Advanced Science, 10(20), 2206713.

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