상품 정보
상품 기본설명
상품 상세설명
General information
Detection significance
The reactive oxygen species produced by the aerobic metabolism in the body can cause the oxidation of DNA, lipid and protein. The secondary reaction of the amino acid side chain of the protein with the lipid oxidation product is the main cause of the formation of the carbonyl. Carbonyl is a biological marker of ROS-mediated protein oxidation.
Detection principle
The content of protein carbonyl increased after oxidation, and the carbonyl group reacted with 2, 4-dinitrophenylhydrazine to form a reddish brown precipitate. The absorbance can be measured at 370 nm after the precipitation is dissolved. The carbonyl content can be calculated indirectly.
The key point
1. When washing the precipitate with anhydrous ethanol-ethyl acetate mixture application solution, the vortex must be voilent the mixing time should not be less than 1 min and the precipitate must be washed to white. If the precipitate still appear yellow, increase the washing times properly of anhydrous ethanol-ethyl acetate mixture application solution to ensure the washing process is sufficient. Otherwise the result will be higher.
2. The speed of centrifuge should not be reduced, otherwise the result will be higher.
3. It is recommended that the round bottom test tube instead of the tip bottom tube should be used to ensure fully washing of the precipitate.
4. The protein content of the samples can’t be determined using the coomassie brilliant blue method.
5. The protein content of the samples should be ranged from 1-10 mg/mL.
Operation procedures
Operation table
| Sample tube | Control tube |
Sample (mL) | 0.1 | 0.1 |
Reagent 3 (mL) | 0.4 |
|
Reagent 4 (mL) |
| 0.4 |
Mix fully by swirling for 1 min, react with shading light at 37℃ for 30 min accurately. | ||
Reagent 5 (mL) | 0.5 | 0.5 |
Mix fully by swirling for 1 min, centrifuge at 13780 g for 10 min at 4℃, discard the supernatant and keep the precipitate. | ||
Anhydrous ethanol-ethyl acetate mixture application solution (mL) | 1.0 | 1.0 |
Mix fully by swirling for 1 min, centrifuge at 13780 g for 10 min at 4℃, discard the supernatant and keep the precipitate. | ||
Anhydrous ethanol-ethyl acetate mixture application solution (mL) | 1.0 | 1.0 |
Mix fully by swirling for 1 min, centrifuge at 13780 g for 10 min at 4℃, discard the supernatant and keep the precipitate. | ||
Anhydrous ethanol-ethyl acetate mixture application solution (mL) | 1.0 | 1.0 |
Mix fully by swirling for 1 min, centrifuge at 13780 g for 10 min at 4℃, discard the supernatant and keep the precipitate. | ||
Anhydrous ethanol-ethyl acetate mixture application solution (mL) | 1.0 | 1.0 |
Mix fully by swirling for 1 min, centrifuge at 13780 g for 10 min at 4℃, discard the supernatant and keep the precipitate. | ||
Reagent 6 (mL) | 1.25 | 1.25 |
Mix fully and incubate at 37℃ water bath for 15 min accurately. | ||
Mix fully by swirling to dissolve the precipitate fully. Centrifuge at 13780 g for 15 min at 4℃, then take the supernatant. Set to zero with reagent 6 and measure the OD values of each tube at 370 nm with 0.5 cm diameter quartz cuvette. Meanwhile, determine the protein concentration of supernatant (E-BC-K318-M, E-BC-K165-S). | ||
Performance characteristics
Technical parameter
| Detection range | 0.02-10 nmol/mgprot | Average inter-assay CV | 8.6% |
| Sensitivity | 0.02 nmol/mgprot | Average intra-assay CV | 4.5% |
| Average recovery rate | 101% |
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