METHOD OF DETERMINATION OF THIOREDOXIN OXIDATIVE MODIFICATION Russian patent published in 2018 - IPC G01N33/53 

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, to biochemical laboratory diagnostics, namely to a method for determining a carbonylated thioredoxin. Method involves incubating 0.25 ml of lysed 1 % triton X-100 cells with 0.5 ml of a 10 mM solution of 2,4-dinitrophenylhydrazine in 2 M HCl, which binds to the carbonyl groups of proteins, incubating the sample at room temperature for 1 hour, adding 0.5 ml of 20 % TCA and centrifuging at 11,000 g for 10 minutes, washing the cake 3 times with 1 ml of a ethanol solution: ethyl acetate (1:1) to extract an excess of 2,4-dinitrophenylhydrazine, that did not react with the carbonyl groups of the oxidized proteins, drying the precipitate and resuspension, further incubating the cell lysate with 0.5 ml of magnetic particles loaded with antibodies to 2,4-dinitrophenol, for 3 hours at 25 °C and continuous mixing. Magnetic particles with the bound antigen-antibody complex are collected by a magnetic support and washed three times with 0.5 ml of 0.01 M sodium phosphate buffer, incubate in a thermostat for 10 minutes at 95 °C, after which they are mixed, then the magnetic particles are collected in a magnetic stand, and the supernatant is used to carry out western blotting with antibodies to thioredoxin.

EFFECT: method for determining the oxidative modification of thioredoxin is proposed.

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