SOD Assay Kit - WST (500 tests)S311_500tests Product Manual

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O₂^.-) into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. In order to determine SOD activity, several direct and indirect methods have been developed. Among these methods, the indirect method using nitroblue tetrazolium (NBT) is commonly used due to the convenience and ease to use. However, there are several disadvantages in the NBT method, such as poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase.
 SOD Assay Kit-WST allows very convenient SOD assay by utilizing Dojindo’s highly water-soluble tetrazolium salt, WST-1 (2-(4-Iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon reduction with the superoxide anion. The rate of the reduction of WST-1 with O₂^.- are linearly related to the xanthine oxidase (XO) activity, and this reduction is inhibited by SOD, as shown in Fig. 1. Therefore, the IC₅₀ (50% inhibition activity of SOD or SOD-like materials) can be determined by the colorimetric method (Patent filing).

Fig.1 Principle of the determination of SOD activity using SOD Assay Kit - WST

WST solution	5 ml x 1
Buffer solution	100 ml x 1
Enzyme solution	100 μl x 1
Dilution buffer	50 ml x 1

Store at 0-5°C. Protect the WST solution and WST working solution from light.

• Plate reader (450 nm filter) 
• 2-20 μl and 20-200 μl pipettes, a multi channel pipette
• 96-well microplate
• Incubator

• For dilution of sample, use Dilution buffer or saline.
• The Enzyme solution is separate into two layers. Therefore, omitting the pipetting process will result in inaccurate experimental results.
• For an accurate measurement, the use of multiple wells per sample is recommended.
• Since superoxide will be released immediately after the addition of Enzyme working solution to a well, use a multichannel pipette to avoid the reaction time lag of each well.

General preparation methods are indicated in our home page. Access www.dojindo.com and go to SOD Assay Kit product information.

For one 96-well plate

WST working solution

Dilute 1 ml of WST solution with 19 ml of Buffer solution.

• WST working solution is stable for 2 months at 0-5 ℃.

Enzyme working solution

Centrifuge the Enzyme solution tube for 5 sec. Mix by pipetting, dilute 15 μl of Enzyme solution with 2.5 ml of Dilution buffer.

• Enzyme working solution is stable at 0-5 ℃ for 3 weeks.

Sample solution

Dilute sample solution with Dilution buffer or saline to prepare sample solution as follows.
　Dilution ratio : 1, 1/5, 1/5², 1/5³, 1/5⁴, 1/5⁵, 1/5⁶

　

Fig. 2 Preparation of sample solution

 

Table 1. Amount of each solution for sample, blank 1, 2 and 3.

	sample	blank1	blank2	blank3
Sample solution	20 μl	-	20 μl	-
ddH2O	-	20 μl	-	20 μl
WST working solution	200 μl	200 μl	200 μl	200 μl
Dilution buffer	-	-	20 μl	20 μl
Enzyme working solution	20 μl	20 μl	-	-

See Table 1, Fig.3 and 4.

1. Add 20 μl of sample solution to each sample and blank 2 well, and add 20 μl of ddH₂O (double distilled water) to each blank 1 and blank 3 well.
2. Add 200 μl of WST working solution to each well, and mix.
3. Add 20 μl of Dilution buffer to each blank 2 and blank 3 well.
4. Add 20 μl of Enzyme working solution to each sample and blank 1 well, and then mix thoroughly.*
5. Incubate the plate at 37℃ for 20 min.
6. Read the absorbance at 450 nm using a microplate reader.
7. Calculate the SOD activity (inhibition rate %) using the following equation.

SOD activity (inhibition rate %) = [(A_{blank 1} - A_{blank 3}) – (A_sample - A_{blank 2})]/ (A_{blank 1} - A_{blank 3}) x 100

• Since superoxide will be released immediately after the addition of Enzyme working solution to a well, use a multi-channel pipette to avoid the reaction time lag of each well.

Inhibition activity can also be determined by a kinetic method. Please determine an incubation time range that has a linearity of the slope before the assay. A good linearity should be observed up to 20 min. For the calculation, use the following equation:

SOD activity(Inhibition rate%) = [(S1 - S3) - (SS - S2)] / (S1 - S3) x 100

S1: slope of blank 1, S2: slope of blank 2, S3: slope of blank 3, SS: slope of sample

『One unit of SOD is defined as the amount of the enzyme in 20 μl of sample solution that inhibits the reduction reaction of WST-1 with superoxide anion by 50%.』

1. Read the dilution ratio at 50% inhibition (IC₅₀) from inhibition curve.
2. Multiply the dilution ratio at IC₅₀ and at the sample preparation to obtain the SOD activity.

Mn-SOD activity can be measured by adding potassium cyanide (final concentration: 1 mmol/l) or diethyldithiocarbamate (final concentration: 1 mmol/l) to the sample solution. These reagents inactivate Cu, Zn-SOD and extracellular-SOD activities.

Table 2 shows compatible concetration of possible interfering materials. If sample contains these materials, please dilute the sample to be below their compatible concentration.
Since 2-mercaptoethanol and dithiothreitol cause a significant increase of the O.D. value, please remove them when sample contains these materials.

Table 2. Compatible concentration of possible interfering materials for SOD assay

Detergents		Solvents		Reducing agents		others
SDS	0.05 %	Ethanol	25 %	Glutathione reduced form	1.25 mmol/l	EDTA	2 mmol/l
Tween 20	0.5 %	DMSO	5 %	Ascorbic acid	0.1 mmol/l	BSA	1 % (w/v)
NP-40	0.5 %