| Usage | Self-supplied consumables:
Microplate reader or visible spectrophotometer (can measure the absorbance at 520 nm)
thermostatic box, ice machine, low temperature centrifuge
96-well plate or microglass cuvette, adjustable pipette gun and tip
deionized water
Homogenizer (if it is a tissue sample)
Reagent preparation:
Ferrous Salt: ready-use type; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
H2O2: ready-to-use type; Before use, balance to room temperature; Store away from light at 4 ° C.
Salicylic Acid: ready-to-use; Before use, balance to room temperature; Store away from light at 4 ° C. Sample preparation:
1. Animal tissues: About 0.1g of sample was weighed, 1 ml of deionized water was added, homogenized in an ice bath, centrifuged at 10,000 g for 10 min at 4 ° C, and the supernatant was taken to be measured.
2. Plant tissue: weigh about 0.1g sample, add 1mL deionized water to mashed, ultrasonic crushing in an ice bath for 5 min (power 20% or 200W, ultrasonic 3 s, interval 7 s, repeated 30 times), centrifuged at 10,000 g at 4℃ for 10 min, and take the supernatant to be tested.
3, cells or bacteria: Collect 5 million cells or bacteria into a centrifuge tube, wash the cells or bacteria with cold PPBS, discard the supernatant after centrifugation, add 1mL deionized water, break the cells or bacteria in an ice bath for 5 min (20% power or 200W, ultrasonic 3 s, interval 7 s, repeated 30 times), and then centrifuge at 10,000 g at 4 ° C for 10 min. The supernatant was taken for testing.
4, serum (plasma) and other liquids with high protein content or turbidity: Take 0.1mL sample, add 1mL deionized water to mix, centrifuge at 10,000 g for 10 min at 4 ° C, and take the supernatant to be tested.
5, honey and urine and other low protein content and clear liquid: direct determination.
6, extracts or drugs: can be prepared into a certain concentration, such as 0.5 mg/mL for determination. Experimental steps:
1, microplate reader or visible spectrophotometer preheat for more than 30 min, adjust the wavelength to 520 nm, visible spectrophotometer with deionized water to zero.
2. Sample determination (successively add the following reagents to 96-well plate or microglass cuvette) : | Reagent | Blank tube（μL） | Standard tube（μL） | Detector tube（μL） | Control tube（μL） | | Ferrous Salt | 40 | 40 | 40 | 40 | | H2O2 | 0 | 40 | 40 | 0 | | Deionized water | 120 | 80 | 40 | 80 | | Salicylic Acid | 40 | 40 | 40 | 40 | | Sample | 0 | 0 | 40 | 40 | The mixture was mixed and the reaction was carried out at 37 ° C for 20 min, and the absorbance at 520 nm was measured. ΔA test =A test -A control, ΔA standard =A standard -A blank (blank and standard Wells only need to be measured once) |
Note: In order to compare the hydroxyl radical scavenging ability of different samples, the same batch of samples must be diluted in the same way, and the extract or drug must be prepared in the same concentration.
The results were calculated as follows:
The calculation formula was: hydroxyl radical clearance D%=(ΔA standard -ΔA measured)÷ΔA standard ×100% |
| Theory | The hydroxyl radical ·OH is the neutral form of the hydroxide ion (OH-) and is highly reactive (easily turning into a hydroxyl group). Hydroxyl radicals act on biological molecules such as proteins, nucleic acids and lipids in the body, causing damage to the structure and function of cells, and then leading to metabolic disorders and diseases. Hydroxyl radical scavenging ability is one of the important indicators of samples of antioxidant capacity, in the study of antioxidant supplements and medicines widely used. Hydroxyl radical scavenging capacity detection kit (trace method) can detect plant and animal tissues, serum (plasma), cells, bacteria, qing, fruit juice, honey, urine samples such as hydroxyl radical scavenging ability, its principle is H2O2 / Fe2 + by Fenton reaction to generate hydroxyl free radicals, Salicylic acid can effectively capture the hydroxyl free radicals reaction with non-ferrous material 2, 3 - dihydroxy benzoic acid, has maximum absorption peak at 520 nm, after joining material with clear ability, non-ferrous material will be reduced, thus can according to the absorbance value of value judgment sample to remove hydroxyl free radical ability. |
| General Notes | 1, different batch number, the composition between different manufacturers don't mix; Otherwise, it may lead to abnormal results.
2, when mixing or redissolving the components, avoid air bubbles.
3, frequently change the suction head to avoid cross contamination between the components.
4, before the experiment, ensure that all the components and equipment are at the right temperature.
5. Keep the samples and all reagents on ice during the assay to avoid denaturation and inactivation.
6, the OD value exceeds 1.3, the sample needs to be diluted.
7, the sample has hemolysis or its own color is dark, need to do self-control. The specific method is to take 200 µ L add 30µ to the diluted sample; L of Extraction Buffer (1x), incubated at 37 ° C for 5 min, the absorbance value at 340 nm was recorded, and the OD value of the self-control was subtracted from the assay Wells for calculation.
8, serum and plasma as well as tissue samples are recommended to be diluted 5 times with Extraction Buffer (1x) for detection.
9, Reagent I and Reagent II were used up and sealed for storage. |
中文
