| Usage |
Self-supplied consumables:
Microplate reader or visible spectrophotometer (can measure the absorbance at 605 nm)
Thermostat, ice machine, low temperature centrifuge
96-well plate or micro-glass cuvette, adjustable pipetting gun and tip
Deionized water
Homogenizer or mortar (if it is a tissue sample)
Reagent preparation:
Reagent Ⅰ : ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
Reagent Ⅱ : ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
ReagentⅢ : ready-to-use; Before use, balance to room temperature; 4 ℃ avoid light preservation.
ReagentⅥ : ready-to-use; Before use, balance to room temperature; 4 ℃ avoid light preservation.
The preparation of the working liquid: before use, the Reagent Ⅳ and Reagent Ⅴ 50:1 to thoroughly incorporated, according to the dosage, active now. If the test sample is the source of mammals, please at 37 ℃ incubation, 5 min. If the samples were other substances, they were incubated at 25 ° C for 5 min.
Sample preparation:
Note: it is recommended to use fresh samples to ensure that the enzyme activity.
The extraction of mitochondrial respiratory chain complexes Ⅱ :
1. Accurately weigh 0.1 g of tissue or collect 5 million cells, add 1 mL ReagentⅠ and 10 µL ReagentⅢ, and homogenize with a homogenizer or mortar ice bath.
2. The homogenate was centrifuged at 600 g for 5 min at 4 ° C, and the supernatant was collected into a new centrifuge tube and the precipitate was discarded.
3. The supernatant was centrifuged again at 11,000 g for 10 min at 4 ° C, and the precipitate was the extracted mitochondria, which was used as the fifth step.
4. (optional) is the cytoplasm supernatant fluid extract, can be used as samples for determination of mitochondria from leakage of mitochondrial respiratory chain complexes Ⅱ (this step is optional, can be used to determine mitochondrial extraction effect).
5. 200 µL ReagentⅡ and 2 µL ReagentⅢ were added to the precipitate, and the precipitate was fully resuspended for further detection of mitochondrial respiratory chain complex Ⅱ enzyme activity.
The experimental steps:
1. The microplate reader or visible spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 605 nm. The visible spectrophotometer was zeroed with deionized water.
2, in 96 - well plates or trace glass color dishes in turn add 10 (including samples, including 25 L L Reagent Ⅵ and 200 (including L working liquid, tapping plate, after thoroughly incorporated, immediately read at 605 nm 0 min initial absorbance value of A1 and A2 absorbance value after 2 min, ΔA=A1-A2 was calculated.
Note: In order to ensure the accuracy of the experimental results, it is necessary to take 1-2 samples for pre-experiment. If the measured absorbance value is too high (greater than 1.5) or ΔA is greater than 0.4, the sample can be diluted by ReagentⅡ before determination. If Δ A small, can increase the numerical sample volume to improve detection is added, if Δ A negative, then samples do not contain complex Ⅱ or degradation.
The results were calculated as follows:
Note: We provide you with the calculation formula, including the derivation process calculation formula and the concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.
A, Calculation formula for determination using 96-well plates
1, according to the fresh weight of the sample
Definition of unit: 1 nmol of 2, 6-dichloroindoxyl consumed per minute per g of tissue in the reaction system was defined as one unit of enzyme activity.
Supernatant of complex Ⅱ dynamic calculation:
Complex Ⅱ on clean energy (U/g fresh weight) = [Δ A1 * V is the total present (epsilon (d) x 10 9] present (W present V sample extraction (V) present T = 1130 x Δ A1 present W
Calculation of complex II activity in precipitation:
Complex Ⅱ precipitation activity (U/g fresh weight) = [Δ A2 * V is the total present (epsilon (d) x 10 9] present sample weight suspended x (W present V V) present T = 226 x Δ A2 present W
Calculation of total activity of sample complex II:
Composite samples Ⅱ total energy is complex in the supernatant Ⅱ vigor and the sum of precipitation in the complex Ⅱ vitality.
Complex Ⅱ total energy (U/g fresh weight) = 1130 * Δ Δ A1 present W + 226 A2 present W
2, according to the cell density calculation
Definition of unit: 1 nmol of 2, 6-dichloroindoxyl consumed per minute per 10 000 cells was defined as one unit of enzyme activity.
Complex Ⅱ activity (U / 10 4 cells) = [Δ A * V is the total present (epsilon (d) x 10 9] present present V (V sample weight suspended x 500) present T = 0.452 x Δ A
V reverse total: total volume of reaction system, 2.25× 10-4 L; ε : 2, 6-dichloroindoxyl molar extinction coefficient, 21×10 3 mol/L/cm; D: 96 light orifice diameter, 0.5 cm; 10 9: unit conversion coefficient, 1 mol=10 9 nmol; V: join the sample volume, 0.01 mL; T: the reaction time, 2 min. Δ A1: supernatant measurements; W: sample weight, g; V extraction: extraction system volume, 1.01 mL; ΔA2: precipitation measurement value; V heavy suspension: suspension precipitation volume, 0.202 mL; 500: total number of cells, 5 million.
B, Calculation formula for determination using a microsized glass cuvette
The above calculation formula of optical path d: 0.5 cm adjustment for d: 1 cm can be calculated.
The results show that:
Experimental examples:
1. 0.1 g of mouse brain tissue was taken for sample processing, followed by the determination steps, and measured with 96-well plates:
ΔA1=A1-A2=0.4268-0.394=0.0328, ΔA2=A1-A2=0.6438-0.56045=0.08335
2. Calculated by sample quality:
Complex Ⅱ on clean energy (U/g fresh weight) = 1130 * Δ A1 present W = 1130 * 0.0328 present 0.1 = 370.64 U/g
Complex Ⅱ precipitation activity (U/g fresh weight)=226×ΔA2÷W=226×0.08335÷0.1=188.371 U/g
The complex Ⅱ fresh weight (U/g) = 1130 * Δ Δ A1 present W + 226 A2 present W = 370.64 + 188.371 = 559.011 U/g |
