Background Introduction:
Plant phenolic substances have the functions of eliminating free radicals, antioxidation and anti-aging. They have high nutritional value and medical health benefits and are widely used in cosmetics, food, medicine and other fields. The detection principle of this kit is that in an alkaline condition, phenolic substances will reduce tungsten molybdate, generating a blue compound. There is a characteristic absorption peak at 760 nm. Measuring the absorbance value at 760 nm can obtain the total phenolic content of the sample. It can detect liquid samples such as plant tissues, fruit juices and honey.
Components：
| P1524795 | Components | Appearance | 48T | 96T | Storage | Quantity Per Test |
| P1524795A | Reagent 1 | Yellow liquid | 2.5 mL | 5 mL | 2-8℃. Store in the dark | 50 uL for 1 reaction |
| P1524795B | Reagent 2 | Colorless clear liquid | 2.5 mL | 5 mL | 2-8℃ | 50 uL for 1 reaction |
| P1524795C | Standard Sample | White powder | 10 mg | 20 mg | RT | Use as per requirements |
Experimental equipment:
Beaker (homogenizer), ice box (ice maker), benchtop centrifuge, adjustable pipette, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tube, microplate reader, 60% ethanol, distilled water (deionized water, ultrapure water are both acceptable).
Index Determination:
It is recommended to select 1-3 samples with significant differences (such as different types or groups) for a preliminary experiment first. This will help you get familiar with the operation process. Based on the results of the preliminary experiment, determine or adjust the sample concentration to prevent unnecessary waste of samples or reagents!
Sample extraction:
- Sample collection: Weigh approximately 0.1g of fresh sample (if the sample is sufficiently moist, increase the sample collection quality); or weigh approximately 0.03g of dried sample (kill the sample at 105℃ for 3 minutes, then dry it at 60℃ until constant weight, grind and sieve through a 40-60 mesh sieve to obtain the dried sample), add 1.5 mL of 60% ethanol (if the fresh sample needs to be ground and homogenized), shake and extract at 60℃ for 2 hours (if volume adjustment is needed, dilute to 1.5 mL with 60% ethanol), at 25℃ at 12000 rpm, centrifuge for 10 minutes, and take the supernatant for testing. [Note]: If the sample quantity is small, reduce the sample quantity proportionally, for example, take 0.02g of dry sample, add 1 mL of 60% ethanol, shake and extract at 60℃ for 2 hours. At 25℃ at 12000 rpm, centrifuge for 10 minutes, take the supernatant, and adjust to 1 mL with 60% ethanol for testing.
- Liquid sample: Directly test; if cloudy, centrifuge and take the supernatant for testing.
- Bacteria/Cultured Cells: First, collect the bacteria or cells into a centrifuge tube, then centrifuge and discard the supernatant; take approximately 5 million bacteria or cells into an EP tube, add 1 mL of 60% ethanol, and perform an 2-hour 60℃ shaking extraction (if for evaporation, dilute to 1 mL with 60% ethanol), at 25℃ at 12000 rpm, centrifuge for 10 minutes, and take the supernatant for testing. [Note]: If increasing the sample volume, you can extract according to the ratio of bacterial or cell quantity (10^4) : extraction liquid volume (mL) as 500:1.
Detection steps:
- Allow the enzyme detector to preheat for 30 minutes (or wait for the instrument to complete its self-check program). Then adjust the wavelength to 760 nm.
- For samples with high total phenol content, such as tea, it is usually necessary to dilute them with distilled water before testing, for example, by diluting 50 times. Alternatively, two samples can be selected for prediction and determination to find out the appropriate dilution factor D for this test.
- Add successively in the 96-well plate：
| Reagent components（µL） | Measurement tube | Blank tube (used only once) |
| Sample | 10 | |
| Reagent 1 | 50 | 50 |
| Mix well. At 25℃ room temperature, let it stand in the dark for 3 minutes. |
| Reagent 2 | 50 | 50 |
| Distilled water | 90 | 100 |
| Mix well, let it stand at room temperature (25℃) for 30 minutes. Transfer all the liquid to a 96-well plate. Measure the absorbance at 760nm (A), and calculate ΔA as A (test tube) - A (blank tube). |
[Note]: 1. If the absorbance value is greater than 1, dilute the sample appropriately before measurement and multiply the calculation formula by the dilution factor D. 2. If ΔA is near zero, increase the sample sampling mass W or increase the sample loading volume V1 (for example, increase it to 30 µL, then reduce the distilled water accordingly to keep the total volume unchanged). Then, the new W and V1 need to be substituted into the calculation formula for re-calculation. 3. If the sample to be tested has a strong background color (such as blue), a sample self-control should be made: replace the reagent with 50 µL distilled water, and follow the same steps as the test tube, then ΔA = A test - A control.
Result calculation:
Standard curve: y = 3.1975x + 0.0451, where x represents the mass of the standard sample (µg) and y represents ΔA.
Total phenol (TP) content (mg/g dry weight) = (ΔA - 0.0451) ÷ 3.1975 × 10⁻³ ÷ (V1 ÷ V × W) × D
Total phenol (TP) content (mg/mg prot) = (ΔA - 0.0451) ÷ 3.1975 × 10⁻³ ÷ (V1 ÷ V × Cpr) × D
Total phenol (TP) content (µg/mL) = (ΔA - 0.0451) ÷ 3.1975 ÷ V1 × D
Total phenol (TP) content (mg/10^4 cells) = (ΔA - 0.0451) ÷ 3.1975 × 10⁻³ ÷ (V1 ÷ V × 500) × D
V --- Add the volume of the extracting liquid;
V1 --- Volume of sample in the reaction, 0.01 mL;
D --- Dilution factor, 1 for undiluted solution;
W---Sample quality, g;
500---Total number of bacteria or cells, in thousands;
Cpr - Protein concentration in the supernatant, mg/mL. It is recommended to use the BCA protein content detection kit of our company.
Appendix: Procedure for preparing the standard curve:
1.The calibration curve is not a mandatory experiment. Users can create the calibration curve according to their experimental needs, or they can directly use the calculation formulas provided in the manual to calculate the results.
2. Prepare the standard stock solution (10mg/mL): Add 1 mL of the extraction solution (60% ethanol) into the standard EP tube, and ultrasonicate to completely dissolve it;
3. Dilute the mother solution with 60% ethanol to obtain six concentration gradients of standard samples, for example: 0, 0.02, 0.04, 0.06, 0.08, 0.1 mg/mL. The concentration of the standard samples can also be adjusted according to the actual sample.
4. The reference table for standard sample dilution is as follows:
| Take 50 uL of the standard solution stock solution, add 4.95 mL of 60% ethanol, and mix well to obtain a 0.1 mg/mL standard dilution solution for later use. | | Standard concentration (mg/mL) | 0 | 0.02 | 0.04 | 0.06 | 0.08 | 0.1 | | Standard diluent (uL) | 0 | 80 | 160 | 240 | 320 | 400 | | 60% ethanol (uL) | 400 | 320 | 240 | 160 | 80 | 0 | | All the standard tubes have been mixed and are ready for use. |
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5.According to the sample addition table of the measuring tube (as shown below), based on the results, subtract the absorbance value at 0 concentration from the absorbance value at each concentration, and then plot a standard curve through the 0 point.
| Reagent components（µL） | Standard tube | 0 concentration tube (used only once) | | Standard Sample | 10 | | | Reagent 1 | 50 | 50 | | Mix well. At 25℃ room temperature, let it stand in the dark for 3 minutes. | | Reagent 2 | 50 | 50 | | Distilled water | 90 | 100 | | Mix well, let it stand at room temperature (25℃) for 30 minutes. Transfer all the liquid to a 96-well plate. Measure the absorbance at 760nm (A), and calculate ΔA as A (test tube) - A (blank tube). |
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Notes:
1. This product is for research use only.
2. For your safety and health, please wear a lab coat and disposable gloves during operation.
