Purified mitochondrial phosphorus to oxygen ratio (ADP/O ) quantitative detection kit
The main purpose
The purified mitochondrial phosphorus-to-oxygen ratio (ADP/O) quantitative detection reagent is a method for determining the decrease of oxygen concentration in fresh living mitochondria in the presence of a known concentration of ADP (III state respiration) by a polarographic system. To analyze the phosphorus to oxygen ratio (P:O ratio) to evaluate the mitochondrial respiratory chain and oxidative phosphorylation coupling degree, that is, the authoritative and classical technical method of energy conversion efficiency. The technology has been carefully developed and successfully tested. It can be used for studies such as mitochondrial physiological functions and drug action mechanisms. The product is strictly sterile, ready to use, easy to operate, in vivo detection, and stable performance.
technical background
Mitochondria are the center of cellular respiratory chain and oxidative phosphorylation. Electron transfer and ATP synthesis are coupled by a proton gradient, that is, the mitochondrial respiratory oxygen is coupled to the phosphorylation of ATP and Pi to synthesize ATP. The ratio of the number of molecules growing ATP (ADP phosphorylation) per gram of atomic oxygen per mitochondria is the ADP/O value. The mitochondrial phosphorus-oxygen ratio directly characterizes the relationship between mitochondrial respiratory oxygen consumption and ATP synthesis. Normal mitochondria have an ADP/O (P/O) of 1.5: a decrease in the ratio of phosphorus to oxygen means mitochondrial uncoupling; an increase in the ratio of phosphorus to oxygen means that mitochondria are well coupled. Its maximum value is 3.3.
product content
Medium solution (Reagent A) ml
Subagent liquid (Reagent B)
Product manual 1 copy
storage method
Stored in a -20 ° C refrigerator, effective for June
User-supplied
CLARK oxygen electrode meter: used to determine dissolved oxygen concentration
Experimental procedure
Before the start of the experiment, fresh mitochondria were prepared and placed in an ice bath. At the same time, the reagent in the -20 °C refrigerator was melted and the oxygen electrode was preheated to 25 °C. Then do the following.
- Add xx ml of media solution (Reagent A) to the reaction glass cell
- Mix thoroughly with micro-magnetic stirring
- Sealing reaction tank
- Start recording oxygen concentration: initial saturated oxygen concentration value is 0.240 micromoles molecular oxygen / ml (25 ° C)
- After 1 minute of continuous recording, add 20 μl of mitochondria to be tested (total 2 mg) (Note: Oxygen concentration may change instantaneously)
- After 1 minute of continuous recording, add xx μl of substrate solution (Reagent B) (note: oxygen concentration begins to decline within 10 seconds; see note 9 )
- Continue recording until there is a linear down slash
- Continue to inject xx microliters of substrate solution (Reagent B) ( Note: oxygen concentration begins to decline within 10 seconds )
- Continue recording until there is a linear down slash
- Calculate the oxygen concentration reduction (micromol/ml):
Calculate ADP/O:
Precautions
- This product is 20 operations
- Wear gloves when handling
- Ensure that the reaction glass tank is clean and sealed to prevent oxygen from flowing in
- Mitochondrial samples must be prepared fresh and are not recommended for cryopreservation; the prepared mitochondrial membrane must be intact and very important; mitochondrial separation kit is recommended - YIJI10006.1
- Avoid air bubbles and air injection when injection is added to the reaction tank
- Avoid ethanol contamination; ethanol increases oxygen concentration; if substrate or inhibitor must be dissolved in ethanol, add 10 μl
- Keep the temperature of the reaction tank constant, the temperature decreases, and the oxygen concentration rises.
- The reaction solution is thoroughly mixed and balanced with ambient oxygen
- Fully freeze-thaw and mix before adding the substrate solution (Reagent B)
- Add reagents strictly according to the regulations, and do not increase the filling reagent capacity.
- The percentage of oxygen concentration is converted to micromoles/ml:
Formula = [measured value (percentage value) ÷ 20.9% (environmental standard oxygen percentage)] X 0.237 (coefficient; micromol / ml )
- The company provides a series of mitochondrial detection reagent products
Quality Standard
- This product has been certified to be stable.
- This product has been identified and sensitive
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