An azocoll (azo-dye impregnated collagen) protease activity assay was performed using a modified protocol described by Jiang et al.16 (link) azocoll (Sigma-Aldrich) was washed with DPBS for 6 h to remove any degraded peptides. The washed azocoll was re-suspended in fresh DPBS at a final concentration of 1.5 mg/mL. Collagenase Type IV (Life Technologies) at a final concentration of 0.1% (weight/volume), azocoll at a final concentration of 0.5 mg/mL, and int-hAM or dev-hAM were incubated for 5 h on an end-to-end rotator at 37°C. Collagenase and azocoll incubated under the same conditions without the tissue samples served as a positive control. azocoll alone served as a negative control. The enzymatic reaction was stopped by centrifugation of the samples at 10,000 g for 8 min. The absorbance of the samples was measured at 550 nm using a spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA).
Azocoll
Manufactured by Merck Group
Sourced in United States
Azocoll is a synthetic substrate used in laboratory settings. It is designed to facilitate the detection and quantification of protease enzyme activity. Azocoll is composed of collagen fibers that have been dyed with a chromogenic azo dye, allowing for colorimetric measurement of protease-catalyzed hydrolysis.
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Lab products found in correlation
Spectramax i3, by Molecular Devices (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Azocoll, by Thermo Fisher Scientific (1 mentions)
Porcine pancreatic elastase, by Merck Group (1 mentions)
Batimastat, by Merck Group (1 mentions)
Bovine pancreatic trypsin, by Merck Group (1 mentions)
Full length murine mmp 12, by R&D Systems (1 mentions)
Bovine pancreatic α chymotrypsin, by Merck Group (1 mentions)
Subtilisin carlsberg, by Merck Group (1 mentions)
Glutathione sepharose 4 fast flow resin, by Cytiva (1 mentions)
Pageruler prestained protein ladder 10 180 kda, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Pgex 6p 1, by Cytiva (1 mentions)
Glutathione sepharose 4 fast flow, by GE Healthcare (1 mentions)
Elastin congo red, by Merck Group (1 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
10 protocols using azocoll
1
Azocoll Protease Activity Assay
2
Quantifying Protease Activities in Samples
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Protease activity in culture media was initially determined with fluorescein isothiocyanate (FITC) casein according to the manufacturer’s instructions and compared to a trypsin standard (Thermo Fisher Scientific, kit # 23266). Values are expressed as units/ml, where one unit is defined as 1.0 mg / 1.0 ml trypsin standard. For determining optimal pH, protease activities were determined with azocoll (Sigma # A4341) added in 5.0 mg portions to 1.0 ml 100 mM potassium phosphate buffer, pH 7.0 at 37°C. Affinity-purified protein samples were added in 200 μl aliquots, the reaction was continued at 37°C for 30 min with gentle shaking, and absorbance was measured at 520 nm [29 (link)].
Serine protease activity of affinity-purified proteins was quantified using a peptide-pNA (Suc-Ala-Ala-Pro-Phe-NHPhNO2) colorimetric substrate (Sigma # S7388; 0.1 mM). Proteinase K (E.C. 3.4.21.64; Sigma # P6556) was used as a positive control to determine relative serine protease activity. Substrate was prepared in 0.1 M Tris-HCl, 0.01 M CaCl2 buffer at pH 7.5. The protein samples (100 μl) were mixed with peptide-pNA substrate (600 μl) and incubated for 10 min at room temperature, then product absorbance was determined at 410 nm and compared to a 4-nitroaniline standard curve. One unit serine protease activity was defined as 1 μmol 4-nitroaniline released per minute at 25°C.
Serine protease activity of affinity-purified proteins was quantified using a peptide-pNA (Suc-Ala-Ala-Pro-Phe-NHPhNO2) colorimetric substrate (Sigma # S7388; 0.1 mM). Proteinase K (E.C. 3.4.21.64; Sigma # P6556) was used as a positive control to determine relative serine protease activity. Substrate was prepared in 0.1 M Tris-HCl, 0.01 M CaCl2 buffer at pH 7.5. The protein samples (100 μl) were mixed with peptide-pNA substrate (600 μl) and incubated for 10 min at room temperature, then product absorbance was determined at 410 nm and compared to a 4-nitroaniline standard curve. One unit serine protease activity was defined as 1 μmol 4-nitroaniline released per minute at 25°C.
3
Bothrops atrox Venom Collagenase Assay
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Bothrops atrox venom collagenase activity was assessed by a colorimetric method adapted from previous studies [33 (link)]. A 0.3% azo-dye impregnated collagen solution (Azocoll, Sigma Aldrich, St.Louis, USA) was prepared with Tris-HCl 0.2 M pH 7.5 as buffer. To each assay tube 200 μL of this solution was added, plus 6 μL of a solution of 1 M CaCl2 (20 mM) and final volume (300 μL) adjusted with distilled water. Venom (50 μg/mL), lapachol and analogues at different concentrations (10–100 μM) were added and its volumes were subtracted from distilled water. Tested substances were dissolved in DMSO and added up to 10 μL to solutions. Tubes were maintained at 37°C for 90 min and stirred gently every 10 min. At the end of this period, tubes were centrifuged at 10,000 rpm for 2 min and the supernatant absorbance was measured on spectrophotometry at an absorbance of 520 nm. Negative controls with pure DMSO were used for each compound and its absorbance subtracted from treatment group.
4
Collagenolytic Activity Assay of Venom
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Azocoll (Sigma, USA) was used to evaluate the collagenolytic activity of venom samples [42 (link)]. Briefly, 50 μL of 5 mg/mL Azocoll solution was incubated with 200 μL venom solutions (125 and 62.5 μg/mL), both in Tyrode buffer, at 37°C for 1 h, under homogenization. The reaction was interrupted by placing samples on ice, and after centrifugation at 5000 g for 3 min, the absorbance of the supernatant was read at 540 nm. One unit of enzymatic activity was defined as the amount of venom that causes an increase of 0.001 units of absorbance per min at 540 nm. Specific activity was expressed as U/min/mg lyophilized venom.
5
Quantifying Collagenolytic Activity in Venom
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Collagenolytic activity was determined as described by Vachova and Moravcova [53 (link)] and modified by Antunes et al. [54 (link)]. 6.25 μg of venom were incubated with 50 μL of 5 mg/mL azocoll (Sigma) solution, both resuspended in Tyrode buffer (137 mM NaCl, 2.7 mM KCl, 3.0 mM NaH2PO4, 10 mM HEPES, 5.6 mM dextrose, 1 mM MgCl2, 2 mM CaCl2, pH 7.4), in Thermo-shaker (Kasvi®) at 37°C and 1000 rpm for 1 h. The reaction was stopped by placing samples on ice. After centrifugation for 3 min at 5,000 x g, the absorbance of the supernatant (200 μL) was measured at 540 nm using a SpectraMax i3 microplate reader (Molecular Devices). One unit of activity was defined as the amount of venom that causes an increase of 0.003 units of absorbance, and specific activity was expressed as U/min/mg of venom. All samples were assayed in triplicate. Data were expressed as mean ±SDM.
6
Quantification of Proteolytic Activity
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For the quantification of the total proteolytic activity present in the total ECP, OMV, and soluble ECP samples, a general proteolytic substrate (Azocoll, Sigma) was employed as previously described (Magariños et al., 1992 (link)). Briefly, 0.02 g of Azocoll was dissolved in 2.5 ml of Tris-Cl 0.1 M pH 7.2, and 100 μl of the sample was added and incubated at 37°C for 1 h. The reactions were stopped by adding 2.5 ml of TCA 10%, centrifuged 2,000 ×g for 15 min at 4°C, and measured using a spectrophotometer (Hitachi U200) at 520 nm. PBS was used as blank. One unit of protease activity (1 U) was defined as the increase of 0.1 in the absorbance value at 520 nm due to azocasein hydrolysis.
7
Collagenolytic Activity Determination
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The determination of the collagenolytic activity was performed according to the methodology described by Wanderley et al. ( 2017) using as substrate Azo dye-impregnated collagen (Azocoll; Sigma Chemical Co., St Louis, MO).
The reaction occurred at 37°C for one hour. After this time, each assay was centrifuged and 1mL of the supernatant was removed for spectrophotometer reading at wavelength at 520nm. One unit of enzyme activity (U) was defined as the amount of enzyme, per milliliter, necessary to increase the absorbance by 0.1.
The reaction occurred at 37°C for one hour. After this time, each assay was centrifuged and 1mL of the supernatant was removed for spectrophotometer reading at wavelength at 520nm. One unit of enzyme activity (U) was defined as the amount of enzyme, per milliliter, necessary to increase the absorbance by 0.1.
8
Azo Dye-Collagen Assay for Enzyme Activity
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Azo dye-impregnated collagen (Azocoll; Sigma Chemical Co., St Louis, MO) assay was carried out according to a modified version of the method developed by Ref. [36] . The absorbance of supernatant was measured at 520 nm by a UV-Vis spectrophotometer (model B582; Micronal, São Paulo, Brazil). One unit of enzyme activity (U) was defined as the amount of enzyme, per milliliter, necessary to increase the absorbance by 0.1, because of the formation of azo dyelinked soluble peptides.
9
Enzymatic Characterization of Proteases
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Human neutrophil elastase, cathepsin G, and α2-macroglobulin were purchased from Athens Research and Technology. Succinyl-A–A–P–F-pNA, batimastat, ecotin, subtilisin Carlsberg, bovine pancreatic trypsin, porcine pancreatic elastase, B. thermoproteolyticus thermolysin, and bovine pancreatic α-chymotrypsin were from Sigma-Aldrich. Serratia sp. serralysin and protealysin, P. aeruginosa LasB and aeruginolysin, F. nucleatum fusolisin, T. denticola dentilysin, and S. gordonii challisin were commercially expressed and purified by Creative Enzymes. Azocoll was from EMD-Millipore, fluorescein-labelled casein (FTC-casein), the PageRuler pre-stained protein ladder (10–180 kDa), restriction endonucleases BamHI and XhoI, and Phusion DNA polymerase were from Thermo Fisher Scientific. The expression vector pGEX-6P-1, glutathione-Sepharose 4 fast flow resin, and 3C (PreScission) protease were from Cytiva. Full-length human MMPs 1, 2, 3, 7, 8, 9, 10, 12, 13, 14, and full-length murine MMP-12 were purchased from R&D Systems Europe. The catalytic domain of human MMP-20 was from Enzo Fischer Scientific. Primers (Table S1† ) were synthesized by Genomed. All other chemical reagents, unless stated otherwise, were from BioShop Canada.
10
Recombinant Protein Expression and Characterization
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The restriction endonucleases BamHI and XhoI, T4 DNA ligase, dNTPs, GeneJET™ Gel Extraction Kit, GeneJET™ PCR Purification Kit, and GeneJET™ Plasmid Miniprep Kit were purchased from Thermo Scientific Fermentas (Vilnius, Lithuania). Phusion DNA Polymerase was obtained from Thermo Scientific Finnzyme (Woburn, MA, USA). The QuikChange Lightning Site-Directed Mutagenesis Kit was obtained from Stratagene (La Jolla, CA, USA). All primers used in the study were synthesized by Genomed and “Pracownia Sekwencjonowania DNA i Syntezy Oligonukleotydów” IBB PAN (Warsaw, Poland). The expression vector pGEX-6P-1, glutathione-Sepharose 4 Fast Flow and 3C protease (PreScission) were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). FTC-casein and Protein Concentrators (9K MWCO, 7 mL) were obtained from Pierce Thermo Fisher Scientific (Rockford, IL). Azocoll was purchased from Calbiochem Merck (Darmstadt, Germany), DQ-gelatin was purchased from Life Technologies Thermo Fisher Scientific (Rockford, IL USA), and Elastin Congo Red was purchased from Sigma (St. Louis, MO). The molecular weight marker: “LMW” (molecular mass range: 14–97 kDa) was purchased from GE Healthcare. Unless otherwise indicated, all other chemicals were obtained from BioShop Canada (Burlington, ON, Canada).
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