Liver Microsomal Preparation

Summary

Liver microsomal preparations, vesicles formed from endoplasmic reticulum fragments obtained by ultracentrifugation of liver tissue hookah, are suitable for biotransformation reactions catalyzed by various metabolic enzymes and inhibition studies. In vitro metabolic experiments were studied by adding different cofactors to the incubation system.

Principle

The basic principle of liver microsome preparation is to study or differentiate in vitro metabolic assays such as cytochrome P450 (CYP), flavin monooxygenase (FMO), and uridine diphosphate glucuronosyltransferases (UGTs) by adding different cofactors to the incubation system.

Appliance

Liver microsomal preparations can be used for research to study in vitro metabolism.

Operation method

Liver Microsomal Preparation

Principle

The basic principle of liver microsome preparation is to study or differentiate in vitro metabolic assays such as cytochrome P450 (CYP), flavin monooxygenase (FMO), and uridine diphosphate glucuronosyltransferases (UGTs) by adding different cofactors to the incubation system.

Materials and Instruments

Equipment:
① internal cut homogenizer, high-speed centrifuge, ultracentrifuge
② several surface dishes, several ice packs
③ crushed ice
④ Beaker, homogenization glass tube
⑤ Stainless steel scissors, (double-beam) ultraviolet spectrophotometer, CO ventilation equipment
Reagents:
① 100 mmol/L potassium phosphate buffer (pH 7.4) with 0.15 mol/L KCI
② 10 mmol/L potassium phosphate buffer (pH 7.4)
③ 2% sodium carbonate solution (prepared with 0.1 mol/L NaOH)
④ 0.5% copper sulfate (prepared with 1% potassium tartrate)
⑤ Bovine serum albumin (BSA) standard solution 500 ug/ml (prepared with 0.1 mol/L NaOH)
⑥ Phenol reagent: Sodium dithionite solution (0.1 g/ml)

Move

The experimental steps for the preparation of liver microsomes were as follows:
A. Tissues were homogenized with 100 mmol/L potassium phosphate buffer (pH 7.4) containing 0.15 mol/L KCI.
B. Centrifuge at 9000×g for 20 minutes, and take the supernatant.
C. Ultracentrifuge at 105,000×g for 60 minutes.
D. Take the precipitate and resuspend it in 100 mmol/L potassium phosphate buffer (pH 7.4), and then centrifuge at 105,000×g for 60 minutes, and take the precipitate, that is, microsomes.
D. Take the precipitate and resuspend it in 100 mmo/L potassium phosphate buffer (pH 7.4), 105000×g ultracentrifuge for 60 minutes, and take the precipitate, i.e. the microsomes.
E. Separate the oily material at the bottom of the human liver microsomes from the protein in the upper layer and store them separately.
F. Resuspend the microsomes in a small amount of potassium phosphate buffer (pH 7.4, 100 mmol/L). G. When it's time for using, take 50 ml of 2% sodium carbonate solution and add 1 ml of 0.5% copper sulfate, and mix well (A solution). (A liquid), put 50 ℃ water bath for 10 minutes (or room temperature for 30 minutes), after cooling, 680 mm colorimetry, calculate the protein content in each 1 ml, dilute each tube of microsomal storage solution into an equal concentration of protein suspension (measurement tube and standard tube are parallel to each other), and establish a standard curve of protein concentration.
H. Dilute microsomal suspension in 0.1 mo/L phosphate buffer (pH 7.4) to 0.5 mg/ml, take 3 ml of each suspension and add 0.5 mg/ml of each suspension to 0.5 mg/ml. H. Dilute the microsomal suspension in 0.1 mo/L phosphate buffer (pH 7.4) to 0.5 mg/ml and add 3 ml of each solution into two colorimetric cups. Scan the UV spectrophotometer in the wavelength range of 500~400 nm and do the baseline correction, and then add 20 ul of sodium dithionite (0.1 g/ml) into the cups.
I. Rapidly fill the tube with CO2 for 30-60 seconds (the filling speed should be slow to prevent foaming), and then scan the tube in the wavelength range of 500-400 nm again after stabilizing it for 5 minutes to obtain the standard curve of reduced cytosolic protein concentration. After stabilizing for 5 minutes, scan again in the 500~400 nm wavelength range to obtain the absorbance (OD) value of reduced cytochrome P450.
J. Calculate the content of cytochrome P450.

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