Protein Carbonyl Colorimetric Assay Kit (for Tissue and Serum Samples)

K119

48T(24S)/ 96T(48S)

The kit should be stored at 2-8℃ shading light for 6 months.

The carbonyl content of the oxidized protein increases, and the carbonyl group can react with 2, 4-dinitrophenylhydrazine to form a reddish-brown precipitate, as shown in the figure. After the precipitate is dissolved, the absorbance value at 370 nm can be read on the spectrophotometer to calculate the carbonyl content of the protein.

When testing the sample, it is necessary to determine the protein concentration, and it is recommended to use BCA Protein Assay Kit (K001).

The kit can be used to detect protein carbonyl in serum, plasma, pleural fluid, cell supernatant and tissue samples.

Microplate reader(360-385 nm), Benchtop centrifuge, Constant temperature water bath/incubator

96-well plate, Precision Micropipettor and clean disposable tips, Clean EP tubes

absolute ethanol, Ethyl acetate, Double distilled water

1.Reagent preparation

①Bring all reagents to room temperature before use. Acid solution should be incubated at 37°C for 10 min before use.

②Preparation of Sulfates(Reagent 2) solution: Take one vial and dissolve it with 3 mL double steaming water. It can be stored for 3 days away from light at 2-8°C.

③Preparation of absolute ethanol and ethyl acetate mixed solution: according to the absolute ethanol: ethyl acetate ratio of 1:1, ready to use.

2.Sample preparation

a.Preparation of sample

Serum and plasma: detect directly.

Tissue samples:

① Harvest the amount of tissue needed for each assay (initial recommendation 20 mg).

② Wash tissue in cold PBS (0.01 M, pH 7.4).

③ Homogenize 20 mg tissue in 180 µL Reagent 1 with a dounce homogenizer at 4℃.

④ Centrifuge at 10000×g for 10 minutes at 4℃ to remove insoluble material. Collect supernatant and keep it on ice for detection.

⑤ Meanwhile, determine the protein concentration of supernatant (K001).

b.Dilution of sample

This kit requires the protein content of the sample to be between 1-10 mg/mL. Before the formal test, 2-3 samples with significant differences should be selected and diluted into different concentrations for pre-test. The recommended dilution factor for different samples is as follows (for reference only):

Note: The diluent is Double distilled water or Reagent 1.

3.Add samples and test

Serum (plasma), pleural fluid, and cell supernatant: The content of protein carbonyl can be measured directly.

Tissue sample: take 0.45mL tissue homogenate, add 0.05mL Sulfates solution (i.e. 9:1 ratio), then the supernatant was placed at room temperature for 10 min, centrifuge 11580 × g at 4°C for 10 min, and the protein carbonyl content was determined.

(1)Control well: Take 0.1 mL sample to be tested and 0.4 mL Reagent 4 into 2 mL EP tube.

Test well: Take 0.1 mL sample to be tested and 0.4 mL Reagent 3 into 2 mL EP tube.

(2)Vortex mixing for 1 min, 37°C away from light for 30 min.

(3)Add 0.5 mL Reagent 5 to each tube, vortex mixing for 1 min, centrifuge 13780 × g at 4°C for 10 min..

(4)Remove the supernatant with a pipette and discard it, retaining the precipitation.

(5)Add 1 mL absolute ethanol and ethyl acetate mixed solution to each tube, vortex mixing for 1 min, centrifuge 13780 × g at 4°C for 10 min, remove the supernatant with a pipette and discard it, retaining the precipitation.(Vortex mixing should not be less than 1 min).

(6)Repeat the previous step 3 times (if there is still yellow residue in the sediment, increase the washing times appropriately).

(7)After washing, each tube was added with 1.25 mL of Reagent 6, vortex mixing, and reacted at 37°C for 15 min.

(8)Vortex mixing to dissolve precipitation, 4°C, 13780 × g, centrifuge for 15 min.

(9) Take 0.3mL of supernatant with micropipette to the enzyme-label plate, and measure OD value at 370 nm on the enzyme-label meter; At the same time, the protein concentration of the supernatant was measured by BCA method.

[Note]

A1 : OD value of Test well

ε : The molar extinction coefficient of carbonyl，22000 L/mol/cm.

d : Optical path, 0.8 cm.

V1 : The volume of the reaction system, 1.25 mL.

V2 : The volume of the sample, 0.1 mL.

Cpr: Concentration of protein in sample, mgprot/mL.

f: Dilution factor of the sample before tested.

1.This assay kit is for Research Use Only. We will not response for any arising problems or legal responsibilities causing by using the kit for clinical diagnosis or other purpose.

2.Please read the instructions carefully and adjust the instruments before the experiments. Please follow the instructions strictly during the experiments.

3.Protection methods must be taken by wearing a lab coat and latex gloves during the experiment.

4.The detection range of the kit is not equivalent to the concentration range of the substance to be measured in the sample. If the concentration of substance is not within the detection range exactly, an extra dilution or concentration should be taken for the sample.

5. It is recommended to take a pre-test if your sample is not listed in the instruction book.

6. The experimental results are closely related to the situation of reagents, operations, environment and so on. FineTest will guarantee the quality of the kits only, and NOT be responsible for the sample consumption caused by using the assay kits. It is better to calculate the possible usage of sample and reserve sufficient samples before use.