ADHP

A stable substrate for peroxidase detection

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5mg

￥2375.00

1900.00

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货号: ajce51140
CAS: 119171-73-2
别名: 10-乙酰基-3,7-二羟基吩嗪,10-Acetyl-3,7-dihydroxyphenoxazine
分子式: C14H11NO4
分子量: 257.24
纯度: >98%
溶解度: DMF: 25 mg/ml,DMSO: 25 mg/ml,DMSO:PBS (pH 7.2)1:5: 0.15 mg/ml,Ethanol: 1.5 mg/ml
储存: Store at -20°C
库存: 现货

Background

10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) is a highly sensitive, stable substrate for horseradish peroxidase (HRP) that enables selective detection of H₂O₂.¹ This colorless, non-fluorescent reagent reacts with H₂O₂ to produce the fluorescent compound resorufin, which can be analyzed using an excitation wavelength of 520-550 nm and an emission wavelength of 585-595 nm. In a 96-well plate format, ADHP enables detection of H₂O₂ at a concentration as low as 5 pmol per 100 ?l sample.¹

1.Zhou, M., Diwu, Z., Panchuk-Voloshina, N., et al.A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidasesAnal. Biochem.253(2)162-168(1997)

Protocol

Kinase experiment:

ADHP, 4-ABAH, 2-ABAH, 4-BAH, 4-FBAH, 4-NBAH, 4-TFMBAH, 3-DMABAH, NaN3 and isoniazid are dissolved in DMSO and subsequently diluted into assay buffer. The final concentration of DMSO in the reaction is less than 0.5 % (v/v), which does not affect fluorescence of the oxidized ADHP product 7-hydroxyl-3H-phenoxazin-3-one (resorufin). Reactions of ADHP (20 μM) are incubated with MPO (2.8 nm) in assay buffer and initiated by the addition of 1/10th volume H2O2 from a serial dilution basin. To determine the effect that the simplest benzoic acid hydrazide inhibitor or its analog 4-TFMBAH has on the heme catalytic ability of MPO, MPO (1.2 μM) is incubated for 10 min with different concentrations of BAH inhibitor (0, 0.025, 0.25, 2.5, 12.5 and 25 mM) with ADHP (40 μM) and timing of the reaction is measured following addition of H2O2 (20 μM) ADHP. All reactions are measured in assay buffer at room temperature. Samples of 20 μL are added to non-reducing sample loading buffers, and then loaded without prior heating and resolved by 4-15% gradient SDS-polyacrylamide gel electrophoresis[1].

参考文献:

[1]. Jiansheng Huang, et al. Ordered Cleavage of Myeloperoxidase Ester Bonds Releases Active site Heme Leading to Inactivation of Myeloperoxidase by Benzoic Acid Hydrazide Analogs. Arch Biochem Biophys. 2014 Apr 15; 548: 74–85.