NB-64-97694-100T | Protein Carbonyl Assay Kit (Microanalysis) Quimigen

Detection Principle

The Protein Carbonyl Content Assay Kit provides a simple and direct method for determining carbonyl content in various biological samples. Carbonyl content is measured through the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form stable dinitrophenylhydrazone (DNP) adducts, which can be detected spectrophotometrically at 370 nm. The absorbance is proportional to the amount of carbonyl groups present.

Packing

Taking 100T/48S packing for example:

Components	Packing	Storage
CB0195M-ES	100 mL x 1	4 °C
CB0195M-A	1 vial (powder) x 5	4 °C, protected from light. * Before use, dissolve each vial with 1 mL of water according to the number of samples. Each vial is sufficient for 10 samples.
CB0195M-B	6 mL x 1	4 °C, protected from light
CB0195M-C	6 mL x 1	4 °C
CB0195M-D	15 mL x 1	4 °C
CB0195M-E	Self-prepared	Mix ethyl acetate and absolute ethanol in equal volumes according to the sample volume.
CB0195M-F	60 mL x 1	4 °C

Note: Before the formal assay, perform a pilot test using 2–3 samples with relatively large expected differences.

Instructions

I. Preparation of Lab Instruments

UV spectrophotometer/plate reader, benchtop centrifuge, adjustable micropipettes, micro quartz cuvettes/96-well plates (UV plates), absolute ethanol, ethyl acetate.

II. Reagent pre-preparation

1.Preparation of tissue samples:

Homogenize the tissue on ice according to the ratio of tissue weight (g) : CB0195M-ES volume (mL) = 1 : 5–10 (it is recommended to weigh about 0.1 g of tissue and add 1 mL CB0195M-ES). Centrifuge at 4000 g, 4 °C for 10 min and collect the supernatant. Add 0.1 mL CB0195M-A, incubate at room temperature for 10 min, then centrifuge at 10000 g, 4 °C for 10 min. Collect the supernatant for assay.

2.Preparation of bacterial or cell samples:
Prepare according to the ratio of cell number (10⁴ cells) : CB0195M-ES volume (mL) = 500–1000 : 1 (it is recommended to add 1 mL CB0195M-ES to 5 × 10⁶ cells).

Disrupt the cells by ultrasonic treatment in an ice bath (power 300 W, 3 s sonication, 7 s interval, total time 3 min). Then centrifuge at 10000 g, 4 °C for 10 min, and collect the supernatant. Keep on ice for testing.

3.Serum and other liquid samples:
Measure directly.

III. Assay Procedure

	Control Tube (μL)	Sample Tube (μL)
Sample	60	60
CB0195M-B		120
CB0195M-C	120
Mix thoroughly and incubate at 37 °C for 1 hour in the dark.
CB0195M-D	150	150
Stand for 5 min, then centrifuge at 4 °C, 12,000 rpm for 15 min. Discard the supernatant and retain the pellet.
CB0195M-E	300	300
Vortex to mix thoroughly, then centrifuge at 4 °C, 12,000 rpm for 10 min. Discard the supernatant and retain the pellet.
CB0195M-E	300	300
Vortex to mix thoroughly, then centrifuge at 4 °C, 12,000 rpm for 10 min. Discard the supernatant and retain the pellet.
CB0195M-E	300	300
Vortex to mix thoroughly, then centrifuge at 4 °C, 12,000 rpm for 10 min. Discard the supernatant and retain the pellet.
CB0195M-F	300	300
Vortex to mix thoroughly, incubate at 37 °C for 15 min, and after the precipitate is completely dissolved, centrifuge at 12,000 rpm for 15 min at 4 °C. Transfer 200 μL of the supernatant into a micro quartz cuvette or a 96-well plate (UV plate). Zero the instrument with CB0195M-F, and measure the absorbance at A370.

Note: The sample tube only needs to be tested once.

IV. Calculation

a. The calculation formula for measurement using a micro quartz cuvette

1.Calculated based on sample protein concentration

Protein carbonyl content (μmol/mg prot) = [ΔA370 × V1 ÷ (ε × d)] ÷ (V2 × C_pr) = 0.227 × ΔA370 ÷ C_pr

2.Calculated based on sample weight

Protein carbonyl content (μmol/g) = [ΔA370 × V1 ÷ (ε × d)] ÷ (W × V2 ÷ V3) = 0.227 × ΔA370 ÷ W

3.Calculated based on cell number

Protein carbonyl content (μmol/10⁴ cells) = [ΔA370 × V1 ÷ (ε × d)] ÷ (Cell number × V2 ÷ V3) = 0.227 × ΔA370 ÷ Cell number

4.Calculated based on liquid volume

Protein carbonyl content (μmol/mL) = [ΔA370 × V1 ÷ (ε × d)] ÷ V2 = 0.227 × ΔA370

Note:

V1: Total volume of the reaction system, 0.3 mL;

ε: Millimolar extinction coefficient of carbonyl groups, 22 L/mmol/cm;

d: Optical path length of the cuvette, 1 cm;

V2: Volume of sample added, 0.06 mL;

V3: Total volume of extraction solution added, 1 mL;

C_pr: Protein concentration of the sample, mg/mL;

W: Sample weight, g.

b. The calculation formula for measurement using a 96-well plate

1.Calculated based on sample protein concentration

Protein carbonyl content (μmol/mg prot) = [ΔA370 × V1 ÷ (ε × d)] ÷ (V2 × C_pr) = 0.454 × ΔA370 ÷ C_pr

2.Calculated based on sample weight

Protein carbonyl content (μmol/g) = [ΔA370 × V1 ÷ (ε × d)] ÷ (W × V2 ÷ V3)= 0.454 × ΔA370 ÷ W

3.Calculated based on cell number

Protein carbonyl content (μmol/10⁴ cells) = [ΔA370 × V1 ÷ (ε × d)] ÷ (Cell number × V2 ÷ V3) = 0.454 × ΔA370 ÷ Cell number

4.Calculated based on liquid volume

Protein carbonyl content (μmol/mL) = [ΔA370 × V1 ÷ (ε × d)] ÷ V2 = 0.454 × ΔA370

Note:

V1: Total volume of the reaction system, 0.3 mL;

ε: Millimolar extinction coefficient of carbonyl groups, 22 L/mmol/cm;

d: Optical path length of the 96-well plate, 0.5 cm;

V2: Volume of sample added, 0.06 mL;

V3: Total volume of extraction solution added, 1 mL;

C_pr: Protein concentration of the sample, mg/mL;

W: Sample weight, g.

Precautions

1.CB0195M-A should be freshly prepared according to the number of samples to be tested before use. After preparation, store at 4 °C. If the solution turns black, it should not be used.

2.CB0195M-B is light-sensitive and easily decomposed upon exposure to light; therefore, the reaction must be performed strictly protected from light.

3.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

4.Please wear a lab coat and disposable gloves.

Protein Carbonyl Assay Kit (Microanalysis)

Cat# NB-64-97694-100T

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