ab65354 Superoxide Dismutase Activity Assay Kit (Colorimetric)
Instructions for Use For the rapid, sensitive and accurate measurement of Superoxide Dismutase Activity in various samples. This product is for research use only and is not intended for diagnostic use. Version 3 Last Updated 18 June 2013
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Table of Contents Table of Contents
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1.
Overview
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2.
Protocol Summary
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3.
Materials Supplied
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4.
Storage and Stability
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5.
Materials Required, Not supplied
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6.
Assay Protocol
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7.
Data Analysis
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8.
Troubleshooting
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1. Overview Superoxide dismutase (SOD) is one of the most important antioxidative enzymes. It catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen. Abcam's Superoxide Dismutase Activity Assay Kit (Colorimetric) is a sensitive kit using WST-1 that produces a water-soluble formazan dye upon reduction with superoxide anion. The rate of the reduction with a superoxide anion is linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD. Therefore, the inhibition activity of SOD can be determined by a colorimetric method.
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2. Protocol Summary Sample Preparation Add Enzyme Working Solution Measure Absorbance
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3. Materials Supplied
Item
Quantity
WST Solution
1 mL
SOD Enzyme Solution
20 μL
SOD Assay Buffer
20 mL
SOD Dilution Buffer
10 mL
4. Storage and Stability Upon arrival, store kit at +4°C. WST WORKING SOLUTION: Dilute the 1 ml of WST solution with 19 ml of Assay Buffer Solution. The diluted solution is stable for up to 2 months at 4°C. ENZYME WORKING SOLUTION: Centrifuge the Enzyme Solution for 5 seconds. Mix well by pipetting – this step is essential as the enzyme has two layers and must be mixed well before dilution –. Dilute 15 μl with 2.5 ml of Dilution Buffer. The diluted enzyme solution is stable for up to 3 weeks at 4°C.
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5. Materials Required, Not supplied
Microcentrifuge
Pipettes and pipette tips
Colorimetric microplate reader
96 well plate
Orbital shaker
Optional: SOD standard
6. Assay Protocol 1. Sample Preparation: a. For blood samples and plasma: Collect blood using citrate or EDTA. Centrifuge at 1,000 x g for 10 min at 4°C. Transfer the plasma layer to a new tube without disturbing the buffy layer and store at -80°C until ready for analysis. Remove the buffy layer from the red cell pellet. Re-suspend the erythrocytes in 5x volume of ice cold distilled water and centrifuge at 10,000 x g for 10 min to pellet the erythrocyte membranes. Store the supernatant at 80°C until ready for analysis. Plasma can be diluted approx. 3-
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10x and the red cell lysate diluted approx. 100x prior to SOD assay. b. For other liquid samples (cell culture media, urine and other biological fluids): use directly in the assay. 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. c. For tissue or cells: Tissue should be perfused with PBS or 150mM KCl to remove any red blood cells. Homogenize tissue or lyse cells in ice cold 0.1M Tris/HCl, pH 7.4 containing 0.5 % Triton X-100, 5mM β-ME, 0.1mg/ml PMSF. Centrifuge the crude tissue homogenate/cell lysate at 14000 x g for 5 minutes at 4°C and discard the cell debris. The supernatant contains total SOD activity from cytosolic and mitochondria. Note: If it is desired to measure SOD activity from cytosol and mitochondria separately, cytosol and mitochondria can be separated
by
using
ab65320
Mitochondria/
Cytosol
Fractionation Kit. SOD activity is then measured from the mitochondria and cytosol fractions separately. 2. Assay Procedure: *Refer to Table 1 for the amount of solution in each well. If you are using a SOD standard (not included with the kit), set up wells for it in the same manner as the sample. a) Add 20 μl of prepared sample to each sample well and Blank 2 well and add 20 μl H2O to each Blank 1 and Blank 3 well.
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b) Add 200 μl of the WST Working Solution to each well. c) Add 20 μl of Dilution Buffer to each Blank 2 and Blank 3 well. d) Add 20 μl of Enzyme Working solution to each sample and Blank 1 well, mix thoroughly. Note: Since the superoxide will be released immediately after the addition of Enzyme Working Solution to each well, use a multiple channel pipette to avoid reaction time lag of each well. e) Incubate plates at 37°C for 20 minutes. f) Read the absorbance at 450 nm using a microplate reader. Table 1. Amount of solution in each well
Sample Solution
ddH2O WST Working Solution Enzyme Working Solution Dilution Buffer
Sample
Blank 1
Blank 2
Blank 3
20 μl
--
20 μl
--
--
20 μl
--
20 μl
200 μl
200 μl
200 μl
200 μl
20 μl
20 μl
--
--
--
--
20 μl
20 μl
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7. Data Analysis Calculate the SOD activity (inhibition rate %) using the following equation: SOD Activity (inhibition rate %)
=
(Ablank1 – Ablank3) – (Asample – Ablank2)
x 100
(Ablank1 – Ablank3)
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8. Troubleshooting Problem
Reason
Solution
Assay not working
Assay buffer at wrong temperature
Assay buffer must not be chilled - needs to be at RT
Protocol step missed Plate read at incorrect wavelength
Unsuitable microtiter plate for assay
Unexpected results
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
Measured at wrong wavelength
Use appropriate reader and filter settings described in datasheet
Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range
Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range
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Samples with inconsistent readings
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
Lower/ Higher readings in samples and standards
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)
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Problem
Reason
Solution
Standard curve is not linear
Not fully thawed kit components
Wait for components to thaw completely and gently mix prior use
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
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 email (
[email protected]) or phone (select “contact us” on www.abcam.com for the phone number for your region).
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Version 3 Last Updated 18 June 2013
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