Carbonyl is an organic functional group formed by carbon and oxygen. Carbonyl groups (aldehydes and ketones) can be introduced into biomolecules through oxidation. The production of carbonyl groups is considered to be the indirect evidence of the oxidation of biomolecules. The measurement of carbonyl content is helpful to the study of physiology and biochemistry.

Carbonyl can react with 2,4-dinitrophenylhydrazine and produce a kind of reddish brown hydrazone compounds, which has a specific absorbance peak at 370 nm. The content of carbonyl can be calculated according to the absorbance value.

The supernatant of sample must be clarified.

1.  Dilution of standard

     Dilute 100 μg/mL standard solution with double distilled water to a serial concentration. The recommended dilution gradient is as follows: 45, 40, 30, 20, 10, 5 μg/mL.

2.  Assay steps:

(1)  Blank tube: add 1.62 mL of double-distilled water into 5 mL EP tube.

      Standard tube: add 1.5 mL of double-distilled water and 0.12 mL of standard with different concentrations into 5 mL EP tube.

      Sample tube: add 1.5 mL of double-distilled water and 0.12 mL of sample into 5 mL EP tube. 

(2)  Add 0.25 mL of Reagent 1 and oscillate fully.

(3)  Stand for 5 min at room temperature. Set the Spectrometer to zero with double-distilled water and measure the OD values of each tube at 370 nm with 0.5 cm diameter cuvette.