MPO activity was determined as previously described39 (link). Briefly, a colon segment (1:10 w/v) in 50 mM phosphate buffer (pH 6.0) was homogenized using an IKA T25 ULTRA-TURRAX basic homogenizer (IKA® Works, Inc., Wilmington, NC) at 4°C. The colon suspension was 10 fold-diluted with 50 mM phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB, Sigma-Aldrich). After sonicating for 10 s and subjecting to three freeze-thaw cycles, each sample was centrifuged at 17,000 x g for 5 min. A 10 μl of each supernatant was then added to 290 μl of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml of o-dianisidine dihydrochlrodie (Sigma) and 0.005% H2O2, and changes in absorbance at 460 nm were measured over 5 min. The protein concentration of the supernatant sample was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). MPO activity was determined by comparison to a standard MPO curve (Sigma-Aldrich).
O dianisidine dihydrochlrodie
Manufactured by Merck Group
O-dianisidine dihydrochloride is a laboratory reagent used in various analytical and diagnostic applications. It is a crystalline compound that serves as a substrate for enzyme-based assays, particularly those involving peroxidase activity. The product can be used to detect and quantify the presence of certain analytes in a sample.
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2 protocols using o dianisidine dihydrochlrodie
1
Quantification of Colon MPO Activity
2
Quantification of Colon MPO Activity
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MPO activity was determined as previously described39 (link). Briefly, a colon segment (1:10 w/v) in 50 mM phosphate buffer (pH 6.0) was homogenized using an IKA T25 ULTRA-TURRAX basic homogenizer (IKA® Works, Inc., Wilmington, NC) at 4°C. The colon suspension was 10 fold-diluted with 50 mM phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB, Sigma-Aldrich). After sonicating for 10 s and subjecting to three freeze-thaw cycles, each sample was centrifuged at 17,000 x g for 5 min. A 10 μl of each supernatant was then added to 290 μl of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml of o-dianisidine dihydrochlrodie (Sigma) and 0.005% H2O2, and changes in absorbance at 460 nm were measured over 5 min. The protein concentration of the supernatant sample was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). MPO activity was determined by comparison to a standard MPO curve (Sigma-Aldrich).
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