ab205810 ADHP HRP substrate Instructions for Use In the presence of H2O2, HRP converts ADHP to the fluorogenic product, resorufin. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 10 July 2015
Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 4 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 5 8. TECHNICAL HINTS 6 ASSAY PREPARATION 9. REAGENT PREPARATION 7 ASSAY PROCEDURE 10. ASSAY PROCEDURE 8 DATA ANALYSIS 11. TYPICAL DATA 9 RESOURCES 12. TROUBLESHOOTING 11 13. NOTES 12 Discover more at www.abcam.com 1
INTRODUCTION 1. BACKGROUND The HRP substrate is a combination of 10-Acetyl-3,7- dihydroxyphenoxazine (ADHP), a sensitive substrate for HRP, and ADHP Dilution Buffer, a stabilized H 2 O 2 solution. In the presence of H 2 O 2, HRP converts ADHP to the fluorogenic product, resorufin. Resorufin is a highly fluorescent molecule, and should be used with excitation filters in the range of 530-540nm, with bandwidths 30nm. The emission filter should be in the range of 590-600nm, with bandwidths 30nm. The signal in the wells should be developed for around 10 minutes. Best results will be obtained if the microplates are developed in the dark, e.g. by covering the microplate with foil. The Stop Solution provided can be used for stopping HRPmediated conversion of ADHP to resorufin. Once added to the wells, signal in the wells is stable for 60 minutes. However, ADHP remaining in the wells is still lightsensitive, and should be handled under low-light conditions until the microplate is read. 2. ASSAY SUMMARY Remove appropriate number of antibody coated well strips. Equilibrate all reagents to room temperature. Prepare all the reagents, standards and samples as instructed. Plates are pre-coated with PTX3. Discover more at www.abcam.com 2
INTRODUCTION Add standard or sample to each well used. Incubate at room temperature. Aspirate and wash each well. Add prepared HRP labeled secondary detector antibody. Incubate at room temperature. Aspirate and wash each well. Add TMB Substrate to each well and incubate. Add Stop Solution at a defined endpoint and record color development. Discover more at www.abcam.com 3
GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY All reagents are stable as supplied at +2-8 C. Do not freeze reagents. Refer to list of materials supplied for storage conditions of individual components. Note the storage conditions for individual prepared components in the Reagent Preparation section. 5. MATERIALS SUPPLIED Item Quantity Storage Condition (Before Preparation) ADHP Dilution Buffer 1 x 15 ml RT ADHP (100X) 1 x 120 µl RT Stop Solution 1 x 2 ml RT Discover more at www.abcam.com 4
GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: 7. LIMITATIONS Exact conditions may vary from assay to assay, a standard curve should be generated for every assay performed. Bacterial or fungal contamination of either samples or reagents or cross-contamination between reagents may cause erroneous results. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh 1X Wash Buffer. Do not allow wells to sit uncovered or dry for extended periods. Avoid exposure of reagents to excessive heat or light during storage and incubation. Optical density values obtained for duplicates should be within 10% of the mean. Duplicate values that differ from the mean by greater than 10% should be considered unreliable and should be repeated. Discover more at www.abcam.com 5
GENERAL INFORMATION 8. TECHNICAL HINTS Avoid preparation or use under bright lights or direct sunlight ADHP is sensitive to bright light. For best results we recommend preparing the Substrate Mix immediately prior to use. It is not stable for long periods once mixed. Kit components should be stored as indicated. All the reagents should be equilibrated to room temperature before use. Samples should be thawed at room temperature. Do not use water baths to thaw samples. Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid crosscontamination. Thoroughly mix the reagents and samples before use by agitation or swirling. All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells. When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells. Many individual components contain preservative. Appropriate protective clothing, glasses and gloves should be worn at all times when handling kit reagents to avoid direct skin contact Discover more at www.abcam.com 6
ASSAY PREPARATION 9. REAGENT PREPARATION Before testing, allow ADHP to thaw prior to use it is solid at 4 C. 9.1 Substrate Mix Dilute ADHP 100-fold with ADHP Dilution Buffer A (e.g. add 20 μl ADHP to 2 ml of the ADHP Dilution Buffer) immediately prior to use. The Substrate Mix is then ready to use. Prepare enough to use 100 µl/well. Discover more at www.abcam.com 7
ASSAY PROCEDURE 10. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. Pipetting Hints: Use different tips to pipette the buffer, standards, samples, and conjugate. Before pipetting each reagent, equilibrate the pipette tip in that reagent (i.e., slowly fill the tip and gently expel the contents, repeat several times). Do not expose the pipette tip to the reagent(s) already in the well. 10.1 Following washing, add 100 µl/well of Substrate Mix. Cover microplate with foil, and incubate for 10 minutes at room temp on a microplate shaker (~300 rpm). 10.2 Add 10 µl/well Stop Solution, and mix for 30 seconds on a microplate shaker. 10.3 Read fluorescence signal with a compatible filter set (e.g. 540/590 nm). Discover more at www.abcam.com 8
DATA ANALYSIS 11. TYPICAL DATA 10 8 RFU (540/590) 10 7 10 6 10 5 10 4 10 3 0.01 0.1 1 10 100 1000 10000 Test Analyte (pm) Figure A Absorbance (450 nm) 1000 100 10 1 0.1 0.01 0.01 0.1 1 10 100 1000 10000 Test Analyte (pm) Figure B Discover more at www.abcam.com 9
DATA ANALYSIS Figure 1. Comparison of ADHP fluorescence (A) and TMB colorimetric (B) assay readouts. Using an ELISA based format, standard curves were generated for a test analyte with concentrations ranging from 40 fm up to 10 nm. After washing away unbound reactants, bound HRP was reacted with ADHP or TMB substrate for 10 mins before stopping each reaction with its respective stop solution. Fluorescence was measured at 540/590 using a FlexStation II 384 (Molecular Devices) and absorbance was measured at 450 nm using an EnVision 2102 Multilabel Reader (Perkin Elmer). Discover more at www.abcam.com 10
RESOURCES 12. TROUBLESHOOTING Problem Possible Causes Solutions Poor Standard Curve No Signal High Background Low sensitivity Standard degraded Curve doesn t fit scale Pipetting Error Plate washings too vigorous Wells dried out Wells are insufficiently washed Contaminated wash buffer Waiting too long to read the plate after adding stop solution Standard is degraded Store and handle standard as recommended Try plotting using different scales Use calibrated pipettes and proper pipetting technique. Check and ensure correct pressure in automatic wash system. Pipette wash buffer gently if washes are done manually. Do not allow wells to dry out. Cover the plate for incubations. Wash wells as per protocol Prepare fresh wash buffer Read plate within 30 minutes Replace standard Mixing or substituting reagents from other kits Avoid mixing components Discover more at www.abcam.com 11
RESOURCES 13. NOTES Discover more at www.abcam.com 12
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