Lactate dehydrogenase is present in all glycolytic cells, and catalyzes the dehydrogenation of lactate to form pyruvate or the reduction of pyruvate to lactate in the presence of NAD+. The purpose of this experiment is to master the principle and method of separating LDH by agarose gel electrophoresis, and to learn to quantitatively determine the relative percentage of each isoenzyme of LDH in animal serum.
Operation method
electrophoretic separation
Principle
Lactate dehydrogenase (LDH) EC ( 1 ,1 , 1 . 2 ) is present in all glycolytic cells. 2) exists in all glycolytic cells, and catalyzes the dehydrogenation of lactate to form pyruvate or the reduction of pyruvate to lactate in the presence of NAD+. The enzyme protein is a tetramer consisting of four subunits, the cardiac type (H-type) and the muscle type (M-type). According to the difference in the proportion of H-type and M-type subunits in the enzyme protein tetramer, LDH isozymes can be classified into five types, i.e., LDH1, LDH2, LDH3, LDH4, and LDH5, which have similar molecular weights of 35,000 subunits but have different charges and therefore different electrophoretic speeds in the electrophoresis. In this experiment, an agarose gel was used as the support medium in pH 8.6 barbiturate buffer. In this experiment, LDH isozymes were separated by electrophoresis in barbiturate buffer at pH 8.6 using agarose gel as support medium. In the presence of oxidized coenzyme I, LDH dehydrogenates lactate to pyruvate and reduces NAD+ to NADH2, which in turn passes hydrogen to phenazine dimethyl sulfate (PMS), which in turn passes hydrogen to nitro-benzotetrazolium blue chloride (NBT) to reduce it to a blue-violet compound. Therefore, the LDH-active zone is blue-violet in color.
Materials and Instruments
Lactate dehydrogenase Move 1 . Preparation of the gel plate: Wash and dry the electrophoresis, seal with tape on both sides, select and mount the comb, adjust the level, and pour the melted 0.5 mm water over the plate. Select and mount the comb, adjust the level, pour the melted 0.7% agarose gel into the plate. Pour the melted 0.7% agarose gel into a suitable amount. Pour a suitable amount of melted 0.7% agarose gel, cool it until it solidifies, and then transfer it to a refrigerator at 4 ℃ for 30~50 min before use. Caveat 1. after LDH isoenzyme color development, often in the LDH1 front edge has a peach color non-specific color region, which is not part of the LDH1, quantitatively should be removed. 2. hemolyzed specimen has obvious effect on the results, can not be used. 3. This method is also applicable to the determination of LDH isoenzymes in various body fluids. For more product details, please visit Aladdin Scientific website.
Agarose Barbiturate Buffer Distilled Water Sodium Lactate Solution Nitrotetrazolium Blue Chloride Aqueous Solution Oxidized Coenzyme I Phenoxydimethyl Sulfate Acetic Acid Solution Aqueous Urea Solution
Electrophoresis tank Electrophoresis apparatus Constant temperature incubator Refrigerator
2 . Spotting and electrophoresis: Add electrophoresis buffer into the tank just to cover the gel. Add 20 μl of serum sample (either in the sample or with bromophenol blue indicator) to the gel with a microsampler, and then add 20 μl of serum sample to the gel at a rate of 4 to 5 mA/cm. ～Add 20 μl of serum sample (in the sample or with bromophenol blue indicator) using a micro-sampler, adjust the current at 4-5 mA/cm gel width, and electrophoresis for about 40-50 min until the serum albumin (or bromophenol blue indicator) is removed. Electrophoresis was stopped when the serum albumin (or bromophenol blue indicator) was 1-2 cm from the end of the gel (if the room temperature was high, electrophoresis was stopped in a refrigerator). If the room temperature is high, electrophoresis should be carried out in a refrigerator.)
3 . Color development: Place the electrophoresed gel in a petri dish with a lid, and spread the gel evenly with a capillary burette. Use a capillary buret to spread a thin layer of colorant evenly on the gel, and then place it in a constant temperature incubator at 37 ℃ for 60 min, so that the same color can be developed. Then place the gel in a constant temperature incubator at 37 ℃ and keep warm for 60 min, so that the isozyme bands can be fully color developed.
4 . Fixation: Put the gel plate after color development into 10% aqueous glacial acetic acid solution and fix it for 10 min. 10 min, pour off the fixative, rinse with distilled water twice, and wash away the excess color solution.
5 . Quantitative analysis: After rinsing the gel plate, cut off the bands with a knife, and transfer them to a plate that has been filled with 3 ml of 25% water. After rinsing the gel plate, cut off each zone with a knife and transfer them into test tubes with 3 ml of 25% urea solution in water, mix them and put them into boiling water for 10 min. After the gel was melted, it was moved to a 37 ℃ water bath and cooled for 10 min before colorimetry was performed. 722 spectrophotometer, wavelength: 1.5 mm. 722 spectrophotometer, wavelength 560 nm, distilled water zero, record the optical density value of each band. The optical density of each band was recorded. The optical density values of each zone were recorded and the percentage of each zone was calculated. The percentage of each band was calculated by dividing the optical density of each band by the sum of the optical densities of each band multiplied by 100%. The percentage of each zone was calculated by dividing the optical density of each zone by the sum of the optical densities of each zone and multiplying by 100%.