ab HRP Substrate Instructions for Use

Transcription

1 ab HRP Substrate Instructions for Use For detecting the activity of many oxidases, related enzymes/substrates or cofactors such as glucose, acetylcholine and cholesterol, L-glutamate, amino acids This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Introduction 3 2. Protocol Summary 4 3. Kit Contents 6 4. Additional Materials Required 6 5. Storage and Handling 6 6. Peroxidase (HRP) Assay Protocol 7 7. H 2 O 2 Assay Protocol 9 8. Troubleshooting 11 2

4 1. Introduction ab HRP Substrate is a sensitive fluorogenic peroxidase substrate that generates a highly red fluorescent product that has maximum absorption of 571 nm and maximum emission of 585 nm. Unlike other HRP substrates such as dihydrofluoresceins and dihydrorhodamines, the air-oxidation of ab is minimal. ab HRP Substrate has been widely used to detect HRP in many immunoassays. It can also be used to detect trace amount of H 2 O 2. H 2 O 2 detection with ab is at least one order of magnitude more sensitive than the commonly used scopoletin assay for H 2 O 2. Because H 2 O 2 is produced in many enzymatic redox reactions, HRP Substrate can be used in coupled enzymatic reactions to detect the activity of many oxidases and/or related enzymes/substrates or cofactors such as glucose, acetylcholine and cholesterol, L-glutamate, amino acids, etc. Spectral Properties: Ex/Em = 571/585 nm ab HRP Substrate can also be used with ab Lysyl Oxidase Activity Assay Kit (Fluorometric). 3

5 2. Protocol Summary Summary for Peroxidase (HRP) Assay One 96-well black Plate Prepare 1X HRP Substrate working solution with 200 µm H 2 O 2 in phosphate buffer (50 μl) Add peroxidase standards(not supplied) or test samples (50 μl) Incubate at room temperature for minutes Monitor the fluorescence intensity at Ex/Em = 540/590 nm Note: This protocol is recommended for peroxidase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs. 4

6 Summary for H 2 O 2 Assay One 96-well Plate Prepare 1X HRP Substrate working solution with 0.4 U/mL peroxidase in phosphate buffer (50 μl) Add H 2 O 2 standards(not supplied) or test samples (50 μl) Incubate at room temperature for minutes Monitor the fluorescence intensity at Ex/Em = 540/590 nm Note: This protocol is recommended for H 2 O 2 assay in solution. The protocol only provides a guideline, should be modified according to the specific needs. 5

7 3. Kit Contents Component Amount HRP Substrate (light sensitive) 1 vial NOTE: Please note that this kit does not contain standards and these have to be provided by researcher. 4. Additional Materials Required DMSO (anhydrous) 200 µm H 2 O 2 in phosphate buffer Black 96 well plate 5. Storage and Handling Keep at -20 C and desiccated. Avoid exposure to light. 6

8 6. Peroxidase (HRP) Assay Protocol Peroxidase (HRP) Assay with HRP Substrate One 96-well black Plate Note: The following is the recommended protocol for peroxidase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs. A. Preparation of HRP Substrate peroxidase working solution: X HRP Substrate stock solution: Add 200 µl of anhydrous DMSO into the vial (mix well). The stock solution should be used promptly; any unused solution should be aliquoted and refrozen at -20 C. Note: Avoid repeated freeze-thaw cycles. Protect from light. 2. 1X HRP Substrate Peroxidase Working Solution: On the day of the experiment, either dissolve HRP Substrate solid in DMSO or thaw an aliquot of the HRP Substrate stock solution at room temperature. Prepare 1X working solution by adding 20 µl of 250X HRP Substrate stock solution (from Step A.1) in 5 ml of 50 mm phosphate buffer or buffer of your choice, ph 7 with 200 µm H 2 O 2. 7

9 Note: HRP Substrate is unstable in the presence of thiols such as DTT and β-mercaptoethanol. Thiols higher than 10 μm (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from: GSH) may interfere with the assay. B. Run peroxidase assay in supernatants: 1. Add 50 μl of 1X HRP Substrate Peroxidase Working Solution (from Step A.2) into each well of peroxidase standard, blank control, and test samples to make the total peroxidase assay volume of 100 μl/well. Note: For a 384-well plate, add 25 μl of sample and 25 μl of 1X HRP Substrate Peroxidase Working Solution into each well. 2. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light. 3. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm. 4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the peroxidase reactions. 8

10 7. H 2 O 2 Assay Protocol H 2 O 2 Assay with HRP Substrate One 96-well black Plate Note: The following is the recommended protocol for peroxidase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs. A. Preparation of HRP Substrate H 2 O 2 working solution: X HRP Substrate stock solution: Add 200 µl of anhydrous DMSO into the vial (mix well). The stock solution should be used promptly; any unused solution should be aliquoted and refrozen at -20 C. Note: Avoid repeated freeze-thaw cycles protect from light. 2. 1X HRP Substrate H 2 O 2 Working Solution: On the day of the experiment, either dissolve HRP Substrate solid in DMSO or thaw an aliquot of the HRP Substrate stock solution at room temperature. Prepare 1X working solution by adding 20 µl of 250X HRP Substrate stock solution (from Step A.1) in 5 ml of 50 mm phosphate buffer or buffer of your choice, ph 7 with 0.4 units/ml peroxidase. 9

11 Note: HRP Substrate is unstable in the presence of thiols such as DTT and β-mercaptoethanol. Thiols higher than 10 μm (final concentration) could significantly decrease the assay dynamic range. NADH and glutathione (reduced from: GSH) may interfere with the assay. B. Run H 2 O 2 assay in supernatants: 1. Add 50 μl of 1X HRP Substrate H 2 O 2 Working Solution (from Step A.2) into each well of H 2 O 2 standard, blank control, and test samples to make the total H 2 O 2 assay volume of 100 μl/well. Note: For a 384-well plate, add 25 μl of sample and 25 μl of 1X HRP Substrate H 2 O 2 Working Solution into each well. 2. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light. 3. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm. 4. The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the H 2 O 2 reactions. 10

12 8. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 11

13 Problem Reason Solution Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349). Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 12

14 For further technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). 13

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16 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 15