For rapid, sensitive and accurate measurement of both Small Molecule Antioxidants and Proteins or Small Molecules alone in various samples.

Transcription

1 ab65329 Total Antioxidant Capacity Assay Kit Instructions for Use For rapid, sensitive and accurate measurement of both Small Molecule Antioxidants and Proteins or Small Molecules alone in various samples. This product is for research use only and is not intended for diagnostic use. Version 6 Last Updated 28 November 2013

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3 Table of Contents 1. Overview 2 2. Protocol Summary 3 3. Materials Supplied 5 4. Storage and Stability 5 5. Materials Required, Not Supplied 6 6. Assay Protocol 6 7. Data Analysis 8 8. Troubleshooting 10 2

4 1. Overview Antioxidants play an important role in preventing the formation of and scavenging of free radicals and other potentially toxic oxidizing species. There are three categories of antioxidant species: enzyme systems (GSH reductase, catalase, peroxidase, etc.), small molecules (ascorbate, uric acid, GSH, vitamin E, etc.) and proteins (albumin, transferrin, etc.). Different antioxidants vary in their reducing power. Trolox is used to standardize antioxidants, with all other antioxidants being measured in Trolox equivalents. Measurement of the combined non-enzymatic antioxidant capacity of biological fluids and other samples provides an indication of the overall capability to counteract reactive oxygen species (ROS), resist oxidative damage and combat oxidative stress-related diseases. In some cases, the antioxidant contribution of proteins is desired whereas in other cases only the contribution of the small molecule antioxidants is needed. Abcam s Total Antioxidant Capacity Assay Kit can measure either the combination of both small molecule antioxidants and proteins or small molecules alone in the presence of our proprietary Protein Mask. Cu 2+ ion is converted to Cu + by both small molecule and protein. 3

5 The Protein Mask prevents Cu 2+ reduction by protein, enabling the analysis of only the small molecule antioxidants. The reduced Cu + ion is chelated with a colorimetric probe giving a broad absorbance peak around 570 nm, proportional to the total antioxidant capacity. 2. Protocol Summary Sample Preparation Standard Curve Preparation Prepare and Add Working Solution Incubate Measure Absorbance 4

6 3. Materials Supplied Item Quantity Cu 2+ Reagent 0.2 ml Assay Diluent 10 ml Protein Mask 10 ml Trolox Standard (1 μmol; Lyophilized) 1 vial 4. Storage and Stability Upon arrival, store kit at +4 C. Cu 2+ REAGENT, ASSAY DILUENT, PROTEIN MASK: Ready to use as supplied and may be kept at room temperature. TROLOX STANDARD: Dissolve the lyophilized Trolox standard in 20 μl of pure DMSO by vortexing, then add 980 μl of distilled water and mix well, generating a 1 mm solution. Following reconstitution, aliquot and store at 20 C. The reconstituted standard is stable for 4 months when stored at 20 C. 5

7 5. Materials Required, Not Supplied DMSO Microcentrifuge Pipettes and pipette tips Colorimetric microplate reader 96 well plate Orbital shaker 6. Assay Protocol 1. Sample Preparation: a) For liquid samples such as urine, culture media, food and drinks: no sample purification from these sources is necessary. If only small molecule TAC is desired, samples should be diluted 1:1 with protein mask. Sample volumes between μl can be assayed per well and should be done in duplicate. You might want to test different sample volumes to find the optimal volume that will give you a reading within the linear range of the standard curve. 6

8 b) For serum samples, we suggest assaying μl without Protein Mask, or 1 10 μl with protein Mask. All well volumes should be adjusted to 100 μl with ddh 2 O. c) For Tissue Samples, Start with mg of the tissue, add μl (or ~4-6 volumes) of the PBS on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Sonication can also be performed instead. Spin down the sample at rpm for 10 min at 4 C and collect the supernatant. Use the supernatant for the subsequent assays. NOTE: The absorbance of samples should be in the linear range of the standard curve (0 20 nmol/well). If they fall outside of this range, they should be re-diluted and re-run. The detection limit of the assay is approximately 0.1 nmole per well (or 1 μm) of Trolox. 2. Standard Curve Preparation: Add 0, 4, 8, 12, 16, 20 μl of the Trolox standard to individual wells. Adjust the total volume to 100 μl with ddh 2 O to give 0, 4, 8, 12, 16, 20 nmol of Trolox standard. 3. Working Solution Preparation: Dilute one part Cu 2+ reagent with 49 parts of Assay Diluent. Dilute enough working solution for the number of assays. Each well requires 100 μl of Cu 2+ working solution. 7

9 4. Add 100 μl Cu 2+ working solution to all standard and sample wells. 5. Cover the plate and incubate at room temperature for 1.5 hours on an orbital shaker. 6. Read the absorbance at 570 nm using the plate reader. 7. Data Analysis Plot standard curve: Plot absorbance at 570 nm as a function of Trolox concentration. Determine sample antioxidant Trolox equivalent concentrations: Sample antioxidant capacity = Sa / Sv = nmol/μl or mm Trolox equivalent Where: Sa is the sample amount (in nmoles) read from the standard curve Sv is the undiluted sample volume added to the wells. 8

10 Top: Quantification of the antioxidant capacity of different antioxidant molecules at 1nmol. Bottom: Trolox standard curve following the kit protocol procedure. 9

11 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 10

12 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) 11

13 Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by (technical@abcam.com) or phone (select contact us on for the phone number for your region). 12

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