Mitochondrial Respiratory Chain Complex I Activity Assay Kit (Microassay)

Self-supplied consumables:
Microplate reader or UV spectrophotometer (can measure the absorbance at 340 nm)
Thermostat, ice machine, low temperature centrifuge
96-well UV plate or microquartz cuvette
Adjustable pipet gun and tip
deionized water
/>Homogenizer or mortar (if it is a tissue sample)

Reagent preparation:
ReagentⅠ : ready-use type; Before use, equilibrate to room temperature; Store at 4 ° C.
ReagentⅡ : ready-to-use; Before use, equilibrate to room temperature; Store at 4 ° C.
ReagentⅢ : ready-to-use; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
Working ReagentⅥ : Prepare before use, 48 T add 1 mL deionized water to fully dissolve, 96 T add 2 mL deionized water to fully dissolve, not used up the dissolved ReagentⅥ please be packed and stored at -20℃ in the dark for 1 month to avoid repeated freezing and thawing. Preparation of
working solution: preparation before use, Reagent Ⅳ and ReagentⅤ were fully mixed at 99:1, according to the dosage, the reagent is used now. If the test sample is of mammalian origin, please incubate at 37℃ for 5 min; If the sample was another species, it was placed at 25 ° C and incubated for 5 min.

Sample preparation:
Note: It is recommended to use fresh samples to ensure enzyme activity.
extraction of mitochondrial respiratory chain complex Ⅰ :
1. 0.1 g tissue was weighed accurately or 5 million cells were collected, and 1 mL ReagentⅠ and 10&micro were added; L Reagent Ⅲ, homogenize with homogenizer or mortar ice bath;
2. The homogenate was centrifuged at 600 g at 4℃ for 5 min. The supernatant was collected into another centrifuge tube and the precipitate was discarded.
3. The supernatant was centrifuged again at 11,000 g for 10 min at 4℃, and the precipitate was the extracted mitochondria, which was used as the fifth step.
4, (optional) the supernatant is the cytosolic extract, which can be used as a sample for the determination of mitochondrial respiratory chain complex Ⅰ leaking from mitochondria (this step is optional, can be used to judge the effect of mitochondrial extraction);
5. Add 200&micro to the precipitate; L ReagentⅡ and 2µ L ReagentⅢ, the precipitate was fully resuspended for further detection of mitochondrial respiratory chain complex Ⅰ enzyme activity.

Procedures:
1. The microplate reader or UV spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 340 nm.
2, in a 96-well UV plate or quartz microcuvette successively add 10 µ L sample, 200 µ L working solution and 15 µ L Working ReagentⅥ, after thorough mixing, immediately read the initial absorbance value A1 at 340 nm at 0 min and the absorbance value A2 after 2 min, and calculate Δ A=A1-A2.
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 (higher than 1.5) or Δ A greater than 0.4, use Reagent Ⅱ diluted samples before determination, Multiply by the dilution factor when calculating the results. If Δ If is small, the detection value can be improved by increasing the sample volume, if Δ If shows negative values, it means that the sample does not contain complex Ⅰ or has been degraded.

result calculation:
Note: we provide you with the calculation formula, including the derivation process calculation formula and concise calculation formula. The two are exactly equal. It is recommended to use the concise calculation formula in bold as the final calculation formula.
A, calculation formula determined using a 96-well UV plate
1, Definition of
units by sample fresh weight: 1 nmol NADH consumed per minute in the reaction system per g of tissue was defined as one unit of enzyme activity. Calculation of mitochondrial respiratory chain complex I activity in
supernatant
complex I activity in the supernatant (U/g fresh weight)=[δA1×V _{reverse total} ÷(ε×d)× 10 9 ]÷ (W÷ V _extract × V _sample )÷ T = 3654 & times; Δ A1÷ Calculation of complex Ⅰ activity of mitochondrial respiratory chain in W
precipitate
complex Ⅰ activity of mitochondrial precipitate (U/g fresh weight)=[δA2×V _{reverse total} ÷(ε× d)×10 9 ]÷ (W÷ V _resuspension × V _-like )÷ T=731× Δ A2÷ Calculation of total complex I activity in W
samples
Total complex I activity is the sum of complex I activity in supernatant and precipitate.
total activity of complex Ⅰ (U/g fresh weight)=3,654× Δ A1÷ W+731× Δ A2÷ W

V _{reverse total} : total volume of reaction system, 2.25× 10-4 L; ε : NADH molar extinction coefficient, 6.22× 10 3 L/mol/cm; d: 96 well UV plate light diameter, 0.5 cm; 10 9: unit conversion coefficient, 1mol=10 9 nmol; V _sample : added sample volume, 0.01 mL; T: reaction time, 2 min; Δ A1: supernatant determination; W: sample weight, g; V _extraction : extraction system volume, 1.01 mL; Δ A2: precipitation determination value; V _resuspension : resuspension precipitation volume, 0.202 mL; 500: total number of cells, 5 million.

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

Results show
Experimental examples:
1, take 0.1 g cabbage sample processing, according to the determination steps, with 96 well UV plate measurement:

Δ A1= A1-a2 =0.7819-0.7804=0.0015, Δ A1= 0.7819-0.7804=0.0015, δ A2=A1-A2=0.783-0.7704=0.0126

was calculated as 3,654×Δ A1÷ W = 3654 & times; 0.0015 & divide; 0.1=54.81 U/g

activity of mitochondrial respiratory chain complex Ⅰ (U/g fresh weight)=731× Δ A2÷ W=731× 0.0126 & divide; 0.1 = 92.106 U/g 

the mitochondrial respiratory chain complexes Ⅰ activity (U/g fresh weight) = 3654 × Δ A1÷ W+731× Δ A2÷ W=54.81+92.106=146.916 U/g

-20℃, stored in the dark, valid for 6 months.