| Usage | Self-supplied consumables:
Microplate reader or visible spectrophotometer (measuring absorbance at 660 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.
ReagentⅡ : 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.
ReagentⅣ : Prepared before use, 48 T was fully dissolved by adding 1 mL deionized water, and 96 T was fully dissolved by adding 2 mL deionized water. Reagents should not use up the repackaging - 20 ℃ avoid light preservation after 1 month, to avoid repeated freezing and thawing.
ReagentⅤ : ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
ReagentⅥ : prepared before use, 48 T was fully dissolved by adding 2 mL deionized water, 96 T was fully dissolved by adding 4 mL deionized water before use. Reagents should not use up the repackaging - 20 ℃ avoid light preservation after 1 month, to avoid repeated freezing and thawing.
ReagentⅦ : Prepared before use, 48 T was fully dissolved by adding 5 mL deionized water, 96 T was fully dissolved by adding 10 mL deionized water before use. Reagents should not use up the repackaging - 20 ℃ avoid light preservation after 1 month, to avoid repeated freezing and thawing.
48 T Reagent Ⅷ : preparation before use, add 5 mL deionized water dissolves, 96 T add 10 mL deionized water dissolves after use. Reagents should not use up the repackaging - 20 ℃ avoid light preservation after 1 month, to avoid repeated freezing and thawing.
Reagent Ⅸ : ready-to-use; Store at room temperature.
Fixed phosphorus Reagent preparation: confecting proportion according to deionized water: Reagent Ⅶ : Reagent Ⅷ : Reagent Ⅸ = 2:1:1: the proportion of 1, with a good set of phosphorus reagents should be light yellow. If it is colorless, the reagent is ineffective; if it is blue, it is phosphorus pollution (please use it as needed).
Note: with reagent had better use the new glass or disposable plastic utensils, to avoid the phosphorus pollution.
Standard curve Settings: press with deionized water as shown in the table will be 10 mM standard dilution of 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0156 mM standard solution.
| NO. | Standard volume（µL） | Deionized water volume（µL） | Standard concentration（mM） | | Std.1 | 100 µL 10 mM | 900 | 0 | | Std.2 | 100 µL of Std.1 (1 mM) | 100 | 0.5 | | Std.3 | 100 µL of Std.2 (0.5 mM) | 100 | 0.25 | | Std.4 | 100 µL of Std.3 (0.25 mM) | 100 | 0.125 | | Std.5 | 100 µL of Std.4 (0.125 mM) | 100 | 0.0625 | | Std.6 | 100 µL of Std.5 (0.0625 mM) | 100 | 0.0313 | | Std.7 | 100 µL of Std.6 (0.0313 mM) | 100 | 0.0156 |
Sample preparation:
Note: The use of fresh samples is recommended to ensure enzyme activity.
The extraction of mitochondrial respiratory chain complexes Ⅴ :
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. Centrifuge the homogenate at 600 g for 5 min at 4℃, collect the supernatant into a new centrifuge tube and discard the precipitate;
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 Ⅴ leaking from mitochondria (this step is optional and can be used to judge the effect of mitochondrial extraction);
5, add 800 in precipitation (including L Reagent Ⅱ and 8 (including L Reagent Ⅲ, fully hung heavy precipitation, for the next step of mitochondrial respiratory chain complexes Ⅴ enzyme activity detection.
Experimental procedures:
1, enzyme standard instrument or visible spectrophotometer preheating above 30 min, adjust to 660 nm wavelength, visible spectrophotometer with deionized water to zero.
2. Enzymatic reaction (add the following reagents in EP tube in turn) :
| Reagent | Blank tube（μL） | Standard tube（μL） | Detector tube（μL） | control tube（μL） | | Reagent Ⅳ | 0 | 0 | 10 | 10 | | ReagentⅤ | 0 | 0 | 40 | 40 | | Sample | 0 | 0 | 50 | 0 | | Blending back cover tightly, 37 ℃ (mammals) or 25 ℃ (other species) accurate incubation for 30 min | | Reagent Ⅵ | 0 | 0 | 20 | 20 | | Sample | 0 | 0 | 0 | 50 |
3, after blending, room temperature (25 ℃ or so), 4000 g, centrifuge for 10 min, take the supernatant.
4, p (in 96 - well plates or trace glass color dish to add the following reagent)
| Reagent | Blank tube（μL） | Standard tube（μL） | Detector tube（μL） | control tube（μL） | | Supernatant | 0 | 0 | 40 | 40 | | Standards | 0 | 40 | 0 | 0 | | Deionized water | 40 | 0 | 0 | 0 | | Fixed phosphorus reagent | 200 | 200 | 200 | 200 |
5. After mixing and standing at room temperature for 10 min, the absorbance value at 660 nm was measured. Computing Δ A = A determination - A contrast, Δ A standard = A standard - A blank. (blank pipe just do 1)
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 (more than 1.5), the sample can be diluted by ReagentⅡ before determination, and the results should be multiplied by the dilution factor. If Δ A small test, sample volume will be increased to join to improve testing values, if negative Δ A test, then samples do not contain complex Ⅴ or degradation. ReagentⅢ has certain toxicity, ReagentⅨ is corrosive, please take good protective measures during operation.
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.
1. Drawing of the standard curve
The concentration of the standard solution was plotted on the Y-axis, and ΔA was plotted on the X-axis to draw the standard curve (concentration on the Y-axis is more convenient to calculate the results).
2, the calculation of inorganic p (Pi) content
As the delta A survey into the equation y values (mM).
3, mitochondrial respiratory chain complexes Ⅴ activity is calculated
(1) Calculated according to fresh weight of sample
Definition of unit: Production of 1 nmol of inorganic phosphorus per minute per g of tissue in the reaction system was defined as one unit of enzyme activity.
The activity of mitochondrial respiratory chain complex Ⅴ supernatant calculation:
Supernatant of complex Ⅴ activity (U/g fresh weight) = (y * 10 V enzymatic qing 6) present present V (V sample extraction x W) present clear present W T = 80.8 x y
The activity of mitochondrial respiratory chain complex Ⅴ precipitation calculation:
Precipitation in the complex Ⅴ activity (U/g fresh weight) = precipitation (y * 10 V enzymatic 6) present present V (V sample weight suspended x W) precipitation present present T = 64.64 x y W
Calculation of total mitochondrial respiratory chain complex V activity in samples:
Samples of the mitochondrial respiratory chain complexes Ⅴ total activity is the supernatant of mitochondrial respiratory chain complexes Ⅴ activity and precipitation in the mitochondrial respiratory chain complexes Ⅴ activity combined.
According to the sample quality calculation:
Mitochondrial respiratory chain complex Ⅴ total activity (U/g fresh weight) =80.8×y supernatant ÷W+64.64×y precipitate ÷W
(2) calculated by cell density
Definition of unit: every 10000 cells per minute produce 1 nmol inorganic phosphorus is defined as a unit of enzyme activity
Mitochondrial respiratory chain complex V activity (U/10 4 cells)=(y×V enzymatic ×10 6)÷(V-like ÷V resuspended ×500)÷T=0.129×y
V enzymatic: enzymatic reaction system volume, 1.2 x 10-4 L; Tendency for 10 6: unit conversion factor, 1 = 10 6 nmol; V-like: adding sample volume, 0.05 mL; T: the reaction time, 30 min; V extraction: extraction system volume, 1.01 mL; W: sample weight, g; V Resuspension: resuspension precipitation volume, 0.808 mL; 500: total number of cells, 5 million.
Experimental examples:
1. Take 0.1 g of green pear tissue for sample processing, follow the determination steps, and measure with a 96-well plate:
ΔA supernatant =A measure-A control =0.404-0.267=0.137, ΔA precipitation =A measure-A control =0.355-0.142=0.213
By putting ∆A supernatant and ΔA precipitate into the equation, y supernatant =0.1518 and y precipitate =0.2358
2. Calculated by sample quality:
Supernatant of complex Ⅴ activity (U/g fresh weight) = 80.8 x y on clear present W = 80.8 x 0.1518 present 0.1 = 122.654 U/g
Complex Ⅴ activity in precipitate (U/g fresh weight)=64.64×y precipitate ÷W=64.64×0.2358÷0.1=152.421 U/g
Then total complex Ⅴ(U/g fresh weight)=80.8×y supernatant ÷W+64.64×y precipitate ÷W=122.654+152.421=275.075 U/g |
| General Notes | 1. Do not mix the components between different batch numbers and different manufacturers; 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, 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 (more than 1.5), the sample can be diluted by ReagentⅡ and then measured, and pay attention to multiply the dilution times when calculating the results.
6, the protein concentration of the sample should be determined by yourself.
7, Reagent Ⅲ has a certain toxicity, Reagent Ⅸ is corrosive, please take good protective measures during operation. |
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