Mitochondrial respiratory chain complex Ⅲ activity detection kit (Micromethod)

Self-supplied consumables:
Microplate reader or visible spectrophotometer (able to measure absorbance at 550 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:
ReagentⅠ : 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; They were stored at 4 ° C in the dark.
ReagentⅥ : ready-to-use; Before use, balance to room temperature; After packaging, it was stored in the dark at -20 ° C for 6 months to avoid repeated freezing and thawing.
Preparation of working solution: Before use, ReagentⅤ was transferred to Reagent Ⅳ, mixed and dissolved, and the surplus reagent was stored at -20℃ in the dark for 2 weeks to avoid repeated freezing and thawing. If the test sample is the source of mammals, please at 37 ℃ for 5 min incubation; If the samples were other species, 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 Ⅲ :
1, accurately say 0.1 g organization or collected 5 million cells, add 1 mL Reagent Ⅰ Reagent and 10 (including L Ⅲ, homogenate dispenser or mortar ice bath homogenate;
2, centrifugal slurry, 600 g, 5 min, 4 ℃, collecting supernatant on to another new centrifugal tube, abandon the precipitation;
3, centrifugal supernatant again, 11000 g, 10 min, 4 ℃, precipitation is the extraction of mitochondria, used as step 5 operation;
4. (optional) is the cytoplasm supernatant fluid extract, can be used as samples for determination of mitochondria from leakage of mitochondrial respiratory chain complexes Ⅲ, 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 Ⅲ enzyme activity.

The experimental steps:
1. Pre-heat the microplate reader or visible spectrophotometer for more than 30 min, adjust the wavelength to 550 nm, and zero the deionized water of visible spectrophotometer.
2. Add 25 µL Reagent Ⅵ and 200 µL working solution to a 96-well plate or micro-glass cuvette, mix thoroughly, react accurately for 2 min, and then add 10 µL sample and mix thoroughly. 550 nm immediately read 0 min initial absorbance value of A1 and A2, absorbance values after 2 min calculation Δ A = A2 - A1.
Note: in order to guarantee the accuracy of experimental results, should be first made preliminary experiment, take one or two samples if Δ A too high (above 1.0), the available Reagent Ⅱ diluted sample measured again after the results note when multiplied by the dilution multiple. If ΔA is small, the detection value can be improved by increasing the sample volume. If ΔA is negative, it means that the sample does not contain complex Ⅲ or has been degraded.

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: The production of 1 nmol of reduced cytochrome C per minute per g of tissue in the reaction system was defined as one unit of enzyme activity.

Calculation of complex III activity in supernatant:

Complex Ⅲ 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 = 1243 x Δ A1 present W

Calculation of the activity of complex Ⅲ in the precipitate:

Complex Ⅲ 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 = 249 x Δ A2 present W

Composite samples Ⅲ total dynamic calculation:

Composite samples Ⅲ total energy is complex in the supernatant Ⅲ vigor and the sum of precipitation in the complex Ⅲ vitality.

Total activity of complex Ⅲ (U/g fresh weight)=1,243×ΔA1÷W+249×ΔA2÷W

2, according to the cell density calculation


Definition of unit: 1 nmol per 10000 cells generated per minute with cytochrome C is defined as a unit of enzyme activity.

Complex Ⅲ 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 = 0.497 x Δ A

V reverse total: total volume of reaction system, 2.35× 10-4 L; Epsilon: reduced cytochrome C molar extinction coefficient, 19.1 x 10 3 mol/L/cm; D: 96 light orifice diameter, 0.5 cm; 10 9: unit conversion coefficient, 1 mol=10 9 nmol; V-like: added 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: the total number of cells, 5 million.

B, Calculation formula for determination using a microsized glass cuvette


The optical diameter d: 0.5 cm in the above calculation formula can be adjusted to d: 1 cm for calculation.

The results show that:

Experimental examples:
1, 0.1 g mice muscle tissue sample processing, according to the measured steps, with 96 orifice measured:

Δ A1 = A2 - A1 = 0.351-0.3413 = 0.0097

Δ A2 = A4, A3 = 0.3516-0.3494 = 0.0022

2. Calculated by sample quality:


Complex Ⅲ on clean energy (U/g fresh weight) = = 1243 x 1243 x Δ A1 present W Δ A1 present W = 1243 x 0.0097 present 0.1 = 120.571 U/g

Complex Ⅲ precipitation activity (U/g fresh weight) = = 249 x 249 x Δ A2 present W Δ A2 present W = 249 x 0.0022 present 0.1 = 5.478 U/g

The complex Ⅲ total energy (U/g fresh weight) = 1243 * Δ Δ A1 present W + 497 A2 present W = 120.571 + 5.478 = 126.049 U/g