Oxidoreductase activity testing method
A test method and reductase technology, applied in the field of medical biological detection, can solve the problems of light color of the solution, easy fading, long reaction time, etc., and achieve the effect of wide application range and convenient operation
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Embodiment 1
[0066] Such as Figure 1 to Figure 3 As shown, the cDNA gene fragment encoding and expressing glutamate dehydrogenase (GDH) protein has a wide range of sources and exists in various living organisms.
[0067] Add 0-8nM GDH, appropriate amount of NADP+, and 5-30mM glutamic acid to 100ul reaction Tris buffer solution (pH7-9) at a final concentration, and shake and react at room temperature for 10-60min. Then, add monosulfonic acid tetrazolium salt detection reagent, after mixing, measure 450nm absorbance value (reference wavelength 620nm), obtain the Escherichia coli source glutamate dehydrogenase linear equation is y=0.21x+0.075 ( 2 =0.999), x is glutamate dehydrogenase activity (nM).
[0068] In the screening test of glutamic acid inhibitors, HJ7 and HJ8 were selected as positive controls. Negative control HJ1. Specific operation: add 50 ul of 0-30 μM inhibitor and 4 nM GDH Tris-NaCl buffer solution (pH 7-9) into a 96-well plate, shake and react for 1-5 hours at room temper...
Embodiment 2
[0070] 1~50mM lactate dehydrogenase corresponding to the base lactate and NAD + , Monosulfonic acid tetrazolium salt detection reagent is mixed in Tris-NaCl buffer or phosphate buffer (pH8~10); the solution to be tested containing lactate dehydrogenase is according to the volume ratio of 1:(0.01~1) Add buffer solution, mix evenly, place at 25-38°C to react for 0.5-4 hours, detect the absorbance at 450nm, and obtain the enzyme activity of the dehydrogenase in the solution to be tested. For details, see Figure 4 .
[0071] The content of lactate dehydrogenase in serum can be used as a detection index for certain diseases. Dilute the serum of different serum samples to an appropriate multiple, and then add 1-50mM lactic acid, NAD + , monosulfonic acid tetrazolium salt detection reagent mixed with Tris-NaCl buffer or phosphate buffer (pH 8 ~ 10); the test solution containing lactate dehydrogenase is added to the buffer according to the volume ratio of 1: (0.01 ~ 1) solution, mi...
Embodiment 3
[0074] 1~50mM Glucose-6-phosphate, NADP + , monosulfonic acid tetrazolium salt detection reagent mixed with Tris-NaCl buffer or phosphate buffer (pH7~8); The volume ratio is added to the buffer solution, mixed evenly, placed at 25-38°C for 0.5-4 hours, and the absorbance at 450nm is detected to obtain the enzyme activity of the dehydrogenase in the solution to be tested. For details, see Figure 5 .
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