Protein Carbonyl Assay Kit (DNPH, Micro Method)

Cat. No.: P1515832

GRADE & PURITY BioReagent

Overview

Protein carbonyls are early markers of protein oxidation modification involving various amino acids. Their level directly indicates the extent of protein oxidative damage and serves as a primary index for assessing protein oxidative injury.

Detection Principle

Carbonyl groups react with 2,4-dinitrophenylhydrazine (DNPH) to form red-colored 2,4-dinitrophenylhydrazone, which exhibits a characteristic absorption peak at 370 nm.

Applicable Samples

Animal/plant tissues, cells, serum, plasma, bacteria.

Reagents, consumables and Equipments not provided

Resource Type	Resource Name	Remarks
Instrument	Microplate Reader	Capable of measuring absorbance at 370 nm
Consumable	96-Well Microplate	UV-transparent plate
Reagent	PBS / Anhydrous Ethanol, Ethyl Acetate	For sample washing / assay use
Others	Homogenizer (for tissue), Incubator, Ice Box, Refrigerated Centrifuge, Adjustable Pipettes and Tips	Using a multichannel pipette can improve efficiency for large sample batches

Precautions

1. Prior to formal testing, it is recommended to perform a preliminary experiment using 2-3 samples with expected significant differences.

2. For tissue and cell samples, normalizing results between samples by measuring protein concentration is recommended. Aladdin B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit are recommended.

3. This kit is compatible with spectrophotometer detection. Please adjust the reagent preparation volumes proportionally according to spectrophotometer cuvette requirements.

4. Biochemical assay reagents generally possess irritant properties or biological toxicity. For your safety and health, please ensure proper biosafety protection throughout the experiment by wearing lab coats, masks, gloves, and head covers, and conduct experiments in a fume hood or biosafety cabinet.

5. This product is for scientific research use only and is not intended for clinical diagnosis.

Protocol (For Reference Only)

1. Reagent Preparation

Reagent Name	Preparation Method	Notes
Extraction Buffer	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C
Working Antioxidant	Prepare immediately before use. Dissolve 0.1 g in 1 mL deionized water. Each 1 mL is sufficient for 10 samples.	Prepare fresh. Toxic, use with caution and proper protection.
Chromogen	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C protected from light. Toxic, use with caution and proper protection.
Reagent D	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C
TCA	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C. Irritating odor; recommended to use in a fume hood.
Guanidine Hydrochloride	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C. Irritating odor; recommended to use in a fume hood.

2. Sample Preparation

*Note: Fresh samples are recommended. If not assayed immediately, samples can be stored at -80°C for up to 1 month.*

2.1 Animal/Plant Tissues

Weigh approximately 0.1 g of tissue sample. Add 1 mL of Extraction Buffer. Homogenize on ice. Centrifuge at 4000 × g for 10 min at 4°C. Collect the supernatant. Add 0.1 mL of Working Antioxidant. Incubate at room temperature for 10 min. Centrifuge at 10000 × g for 10 min at 4°C. Collect the supernatant and keep on ice for testing.

2.2 Cell or Bacterial Samples

Collect 5 × 10⁶ cells. Wash the cells or bacteria with cold PBS. Centrifuge at 800 × g for 2 min and discard the supernatant. Add 1 mL of Extraction Buffer. Sonicate on ice for 5 min (20% power or 200 W, 3 s on, 7 s off, repeat 30 times). Centrifuge at 10000 × g for 10 min at 4°C. Collect the supernatant and keep on ice for testing.

2.3 Serum (Plasma)

Test directly.

3. Assay Procedure

3.1 Microplate Reader Setup

Preheat the microplate reader for at least 30 min. Set the wavelength to 370 nm.

3.2 Reaction System Setup

Prepare the reaction in 1.5 mL microcentrifuge tubes as described below. Each test sample requires a corresponding control tube.

Reagent	Control Tube (μL)	Test Tube (μL)
Sample	60	60
Chromogen	0	120
Reagent D	120	0

Mix thoroughly. Incubate at 37°C for 1 h in the dark.

TCA

150

150

Let stand for 5 min. Centrifuge at 12000 × g for 15 min at 4°C. Discard supernatant, retain pellet.

Anhydrous Ethanol	150	150
Ethyl Acetate	150	150

Vortex mix thoroughly. Centrifuge at 12000 × g for 15 min at 4°C. Discard supernatant, retain pellet. Repeat this wash step 3 times.

Guanidine Hydrochloride

300

300

Vortex mix thoroughly. Incubate at 37°C for 15 min until the pellet is completely dissolved. Centrifuge at 12000 × g for 15 min at 4°C.

3.3 Absorbance Measurement

Transfer 200 μL of the supernatant to a UV-transparent 96-well plate. Measure the absorbance at 370 nm. Record the values as A_Control and A_Test.

4. Calculation of Results

4.1 Data Processing

Calculate ΔA = A_Test - A_Control.

4.2 Calculation of Sample Protein Carbonyl Content

Based on Sample Mass

Protein Carbonyl Content (μmol/g) = [ΔA × V_RTotal ÷ (ε × d)] ÷ (W × V_S ÷ V_STotal) × n = 0.454 × ΔA ÷ W × n

Based on Cell or Bacterial Count

Protein Carbonyl Content (μmol/10⁴ cells) = [ΔA × V_RTotal ÷ (ε × d)] ÷ (N × V_S ÷ V_STotal) × n = 0.454 × ΔA ÷ N × n

Based on Liquid Volume

Protein Carbonyl Content (μmol/mL) = [ΔA × V_RTotal ÷ (ε × d)] ÷ V_S × n = 0.454 × ΔA × n

Based on Protein Concentration

Protein Carbonyl Content (μmol/mg prot) = [ΔA × V_RTotal ÷ (ε × d)] ÷ (V_S × Cpr) × n = 0.454 × ΔA ÷ Cpr × n

Parameter Definitions:

V_RTotal: Total volume of the reaction system (0.3 mL)

ε: Molar extinction coefficient of carbonyl (22 mL/μmol/cm)

d: Light path of the 96-well plate (0.5 cm)

V_S: Volume of sample added (0.06 mL)

V_STotal: Volume of Extraction Buffer added for homogenization (1 mL)

Cpr: Protein concentration of the sample (mg/mL)

W: Sample mass (g)

N: Cell or bacterial count (unit: 10⁴ cells. Example: for 5 × 10⁶ cells, N = 500)

n: Sample dilution factor.

Example of Results

Mouse kidney (0.1 g) was processed according to the protocol and measured using a UV-transparent 96-well plate. The measured values were ΔA = A_Test - A_Control = 0.103 - 0.071 = 0.032.

Calculation based on sample mass:

Protein Carbonyl Content (μmol/g) = [ΔA × V_RTotal ÷ (ε × d)] ÷ (W × V_S ÷ V_STotal) × n = 0.454 × ΔA ÷ W × n = 0.145 μmol/g.

Frequently Asked Questions (FAQ)

Q: What should I do if the measured ΔA of the sample is too high or too low?

A: If ΔA > 0.8, it indicates the protein carbonyl content in the sample is too high. The sample should be appropriately diluted with Extraction Buffer (multiply by the corresponding dilution factor in the calculation formula) or the amount of starting sample material should be reduced. If ΔA < 0.005, the amount of starting sample material can be appropriately increased.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (6 months). Store in the dark.

Contents & Storage

P1515832	Components	Appearance	48T/48S	96T/96S	Storage
P1515832A	Extraction Buffer	Liquid	60 mL	120 mL	2-8℃.
P1515832B	Antioxidant	Powder	1 EA (48T)	1 EA (96T)	2-8℃. Store in the dark.
P1515832C	Chromogen	Liquid	7.5 mL	15 mL	2-8℃. Store in the dark.
P1515832D	Reagent D	Liquid	7.5 mL	15 mL	2-8℃.
P1515832E	TCA	Liquid	18 mL	36 mL	2-8℃.
P1515832F	Guanidine Hydrochloride	Liquid	36 mL	72 mL	2-8℃.

Please check the volume of each component before starting the experiment.

An additional 10% volume is provided beyond the specified kit size for standard curve preparation or preliminary experiments.