It can be ground in liquid nitrogen and other steps should be basically the same.
1, materials and methods
The separated liquid of heart, liver, muscle, brain and serum of crucian carp after centrifugation was analyzed by electrophoresis.
Electrophoresis uses polyacrylamide as a support, and the concentrations of concentrated gel and separated gel are shown in Table 2.
Name of reagent: dosage of each reagent required for preparing 20ml separation gel with different concentrations/dosage of reagent required for preparing 10ml concentrated gel.
5% 7.5% 10% 15% 3%
Separation glue storage 3.33 5.00 6.66 10.00
Separation gel buffer 2.50 6.50 2.50 2.50
Concentrated glue storage solution 1.65
Concentrated gel buffer 1.25
1% TEMED 2.00 2.00 2.00 2.00 1.00
Distilled water 12.07 6.50 8.74 5.40 7.03
After mixing, put it into a vacuum dryer and pump air 10 minute.
1% AP 0. 10 0. 10 0. 10 0. 10 0.05
Table 2 Gel Preparation of Discontinuous Systems
In order to prevent LDH and its isoenzymes from being affected by gel polymerization residues (such as AP). ) and cause enzyme inactivation or other human influence, should be pre-electrophoresis at 20mA for half an hour before adding samples. Then, turn off the power supply, add five kinds of tissue serum of heart, liver, muscle, brain and serum and the same volume of sucrose-bromophenol blue mixed solution, and pay attention to the sampling order. Electrophoresis was carried out with Tris- glycine electrode buffer at 20mA for 4 hours. After electrophoresis, take off the rubber plate, put it into active dyeing solution for dyeing, and then rinse it with water. The preparation of reactive dye solution is shown in Table 3.
Storage solution NAD+ sodium lactate NaCl NBT PMS phosphate buffer solution
Dose/ml
Table 3 preparation of LDH active dye solution