ADHP(Amplex Red)
(Synonyms: 过氧化氢探针) 目录号 : GB30275
测量过氧化氢浓度或过氧化物酶活性。
Cas No.:119171-73-2
Sample solution is provided at 25 µL, 10mM.
在辣根过氧化物酶(HRP)存在下,ADHP与H2O2以1:1的化学计量比反应,生成高荧光的Resorufin。 Resorufin产物可通过荧光微板读取器轻松读取,其激发波长为530-560nm,发射波长为590nm。荧光值与样品中H2O2或过氧化物酶水平成比例。通过与相应标准曲线的比较,可以确定未知样品中的H2O2或过氧化物酶含量。
Storage:
Upon receipt, aliquot and store the ADHP probe and HRP at -20ºC. Avoid multiple freeze/thaw cycles. Store the remaining kit components at 4ºC. ADHP is light sensitive, must be stored accordingly.
Preparation of Reagents
Note: All reagents must be brought to room temperature prior to use.
• 1X Assay Buffer: 50 mM Tris–HCl buffer pH7.4
• ADHP stock solution:a 10 mM solution in DMSO.
• HRP stock solution: a 100 U/mL solution in glycerol*.
• H2O2 stock solution: an 8.8 M solution
*Note: One unit is defined as the amount of enzyme that will form 1.0 mg purpurogallin from pyrogallol in 20 seconds at pH 6.0 and 20ºC.
• ADHP/HRP Working Solution (Hydrogen Peroxide Assay): If measuring hydrogen peroxide, prepare an ADHP/HRP Working Solution by adding ADHP to a final concentration of 100 µM and HRP to a final concentration of 0.2 U/mL in 1X Assay Buffer (eg. Add 50 µL ADHP stock solution and 10 µL HRP stock solution to 4.940 mL 1X Assay Buffer). This volume is enough for ~100 assays. The ADHP/HRP Working Solution is stable for 1 day. Prepare only enough for immediate use.
• ADHP/H2O2 Working Solution (Peroxidase Assay): If measuring peroxidases, prepare the ADHP/H2O2 Working Solution by adding ADHP to a final concentration of 100 µM and H2O2 to a final concentration of 2 mM in 1X Assay Buffer. First perform a 1:1000 dilution of the stock H2O2 in 1X Assay Buffer. Use only enough for immediate applications (eg. Add 5 μL of H2O2 to 4.995 mL 1X Assay Buffer). This solution has a concentration of 8.8 mM. Use this 8.8 mM H2O2 solution to prepare a 2 mM H2O2 solution in ADHP/1X Assay Buffer (eg. Add 50 µL ADHP stock solution and 1.14 mL of the prepared 8.8 mM H2O2 solution to 3.81 mL 1X Assay Buffer). This volume is enough for ~100 assays. The Working Solution is stable for 1 day. Prepare only enough for immediate use.
Preparation of Samples
• Cell culture supernatant: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Prepare the H2O2 standard curve in the same non-conditioned media. Serum should be avoided, as it interferes with the assay. Note: Maintain pH between 7 and 8 for optimal working conditions as the ADHP is unstable at high pH (>8.5).
• Cell lysate: Resuspend cells at 1-2 x 106 cells/mL in PBS or 1X Assay Buffer. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates can be assayed undiluted or titrated as necessary.
• Plasma, Serum or Urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Undiluted serum or plasma may interfere with the assay.
Notes:
• All samples should be assayed immediately or stored at -80°C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator. Always run a standard curve with samples.
• A serial dilution will be necessary depending on the total H2O2 or peroxidase present. Extremely high levels of H2O2 (≥ 500 µM final concentration) or peroxidase (≥ 100 mU/mL) can lower the fluorescence because excess H2O2 or peroxidase can further oxidize the reaction product, Resorufin, to nonfluorescent product Resazurin.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the ADHP probe and could result in erroneous readings. To minimize this interference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a final concentration of 40 U/mL .
• Avoid samples containing DTT or β-mercaptoethanol since Resorufin is not stable in the presence of thiols (above 10 μM).
Preparation of Standard Curves
• H2O2 Standard: To prepare the H2O2 standards, first perform a 1:1000 dilution of the stock H2O2 in 1X Assay Buffer. Prepare only enough for immediate use (e.g. Add 5 μL of H2O2 to 4.995 mL 1X Assay Buffer). This solution has a concentration of 8.8 mM. Use this 8.8 mM H2O2 solution to prepare standards in the concentration range of 0 µM – 100 µM by further diluting in 1X Assay Buffer (e.g. Add 11.5 µL of H2O2 to 988.5 µL 1X Assay Buffer - see Table 1 below). H2O2 diluted solutions and standards should be prepared fresh.
• Peroxidase Standard: To prepare the peroxidase standards, first perform a 1:1000 dilution of the stock HRP in 1X Assay Buffer (e.g. Add 5 μL of HRP stock to 4.995 mL 1X Assay Buffer). Prepare only enough for immediate use. This solution has a concentration of 100 mU/mL. Use this 100 mU/mL solution to prepare standards in the concentration range of 0 mU/mL – 10 mU/mL by further diluting in 1X Assay Buffer (see Table 2 below). HRP diluted solutions and standards should be prepared fresh.
Assay Protocol
I. Hydrogen Peroxide
1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 µL of each sample (H2O2 standard, control or unknown) into an individual microtiter plate well.
3. Add 50 µL of ADHP/HRP Working Solution to each well. Mix the well contents thoroughly and incubate for 30 minutes at room temperature protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the kinetics of the reactions.
4. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.
5. Calculate the concentration of peroxide within samples by comparing the sample RFU to the standard curve. Subtract the value from the zero H2O2 control.
II. Peroxidase
1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 µL of each sample (HRP standard, control or unknown) into an individual microtiter plate well.
3. Add 50 µL of ADHP/ H2O2 Working Solution to each well. Mix the well contents thoroughly and incubate for 30 minutes at room temperature protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the kinetics of the reactions.
4. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.
5. Calculate the concentration of peroxidase within samples by comparing the sample RFU to the standard curve. Subtract the value from the zero HRP control.
This protocol only provides a guideline, and should be modified according to your specific needs.
Cas No. | 119171-73-2 | SDF | |
别名 | 过氧化氢探针 | ||
分子式 | C14H11NO4 | 分子量 | 257.24 |
溶解度 | DMSO : 125 mg/mL (485.93 mM; Need ultrasonic) | 储存条件 | Store at -20°C, protect from light |
General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液;一旦配成溶液,请分装保存,避免反复冻融造成的产品失效。 储备液的保存方式和期限:-80°C 储存时,请在 6 个月内使用,-20°C 储存时,请在 1 个月内使用。 为了提高溶解度,请将管子加热至37℃,然后在超声波浴中震荡一段时间。 |
||
Shipping Condition | 评估样品解决方案:配备蓝冰进行发货。所有其他可用尺寸:配备RT,或根据请求配备蓝冰。 |
制备储备液 | |||
![]() |
1 mg | 5 mg | 10 mg |
1 mM | 3.8874 mL | 19.4371 mL | 38.8742 mL |
5 mM | 0.7775 mL | 3.8874 mL | 7.7748 mL |
10 mM | 0.3887 mL | 1.9437 mL | 3.8874 mL |
第一步:请输入基本实验信息(考虑到实验过程中的损耗,建议多配一只动物的药量) | ||||||||||
给药剂量 | mg/kg | 动物平均体重 | g | 每只动物给药体积 | ul | 动物数量 | 只 | |||
第二步:请输入动物体内配方组成(配方适用于不溶于水的药物;不同批次药物配方比例不同,请联系GLPBIO为您提供正确的澄清溶液配方) | ||||||||||
% DMSO % % Tween 80 % saline | ||||||||||
计算重置 |
计算结果:
工作液浓度: mg/ml;
DMSO母液配制方法: mg 药物溶于 μL DMSO溶液(母液浓度 mg/mL,
体内配方配制方法:取 μL DMSO母液,加入 μL PEG300,混匀澄清后加入μL Tween 80,混匀澄清后加入 μL saline,混匀澄清。
1. 首先保证母液是澄清的;
2.
一定要按照顺序依次将溶剂加入,进行下一步操作之前必须保证上一步操作得到的是澄清的溶液,可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet