Alkaline protease activity was tested using a modified method previously described (40 (link)). Samples containing 1 mg of Hide powder azure (Sigma) dissolved in buffer (0.075 mL) consisting of 20 mM Tris/HCl, pH 8.0 and 1 mM CaCl2 were mixed with 0.025 mL of the culture supernatants. The reaction mixtures (0.1 mL) were incubated at 37 °C for 1 h. Undissolved substrate was removed via centrifugation at 4000 × g for 5 min. The absorbance of the reaction mixtures was determined at 595 nm on a SpectraMax M Series Multi-Mode Microplate Reader (Molecular Devices). Protease activity was expressed in terms of protease units per milliliter (U/mL), where one unit is equivalent to an increase in OD595 of 1.0 per hour at 37 °C. Protease activity was normalized to account for variation in bacterial cell growth based on the OD600 of the culture at 5 h. Relative changes were calculated based on the protease activity in medium alone.
Hide powder azure
Manufactured by Merck Group
Sourced in United States
Hide powder azure is a fine, blue-colored powder derived from animal hides. It is used as a colorant or pigment in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m series multi mode microplate reader, by Molecular Devices (2 mentions)
Chromogenic substrates, by Werfen (1 mentions)
Bovine fibrinogen, by Merck Group (1 mentions)
Casein, by Merck Group (1 mentions)
Keratin azure, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Elastin congo red, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cysteine, by Merck Group (1 mentions)
5 protocols using hide powder azure
1
Alkaline Protease Activity Measurement
2
Enzymatic Activity Characterization Protocol
3
Alkaline Protease Activity Measurement
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Alkaline protease activity was tested using a modified method previously described (40 (link)). Samples containing 1 mg of Hide powder azure (Sigma) dissolved in buffer (0.075 mL) consisting of 20 mM Tris/HCl, pH 8.0 and 1 mM CaCl2 were mixed with 0.025 mL of the culture supernatants. The reaction mixtures (0.1 mL) were incubated at 37 °C for 1 h. Undissolved substrate was removed via centrifugation at 4000 × g for 5 min. The absorbance of the reaction mixtures was determined at 595 nm on a SpectraMax M Series Multi-Mode Microplate Reader (Molecular Devices). Protease activity was expressed in terms of protease units per milliliter (U/mL), where one unit is equivalent to an increase in OD595 of 1.0 per hour at 37 °C. Protease activity was normalized to account for variation in bacterial cell growth based on the OD600 of the culture at 5 h. Relative changes were calculated based on the protease activity in medium alone.
4
Optimized Protease Activity Assay for P. fluorescens
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The Hide powder azure assay was used for the detection of the protease quantity from P. fluorescens (Himelbloom, and Hassan 1985 (link)). Hide powder azure was obtained from Sigma and the procedure was modified for protease assay. The enzyme assay procedure was optimized by varying the amount of Hide powder (0.01, 0.02, and 0.03 g), rotation (static, 2 rpm, and 10 rpm), filtration (no filtration, and with 0.47µ), and pH values (4.5, 5.5, 6.5, 7.0, 7.5, 8.5, 1nd 9.0). The enzymatic reaction was carried out in duplicate by adding 3 mg of hide powder into 4.0 mL of the supernatant at pH 7.1 into a 5 mL vial. The vial was agitated and then incubated for 1.5 h at 37 °C in a water bath. Each vial was centrifuged at 10,000 rpm for 10 min and the supernatant was removed carefully and then passed through a Millipore filter pump. Absorbance at 600 nm of the filtrate was determined against the control. Controls contained everything except the supernatant. Protease activities unite was defined as absorbance at 600 per CFU/mL and measured according to our following designed equation:
All protease activity values are multiplied by a value of (10-8).
All protease activity values are multiplied by a value of (10-8).
5
Biochemical Assay Materials Analysis
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Casein (from bovine milk), Coomassie brilliant blue G-250, cysteine, bovine serum albumin, Elastin-Congo Red, Hide Powder Azure, Keratin Azure, and Tris were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Ethylenediaminetetraacetic acid was purchased from Invitrogen (Carlsbad, California, USA), sodium phosphate (98%) from Carlo Erba (Rodano, MI, Italy), detergent Azymol 6SE from Pellital (Victoria, BA, Argentina). All other chemicals were of analytical grade.
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