Succinate Dehydrogenase (SDH) Colorimetric Assay Kit

K142

96T (94 samples)

0.83U/L

0.83-65.42U/L

To assay the activity of succinate dehydrogenase (SDH) in animal tissues and cell samples.

Store at-20°C in a dark for 6 months.

Succinate dehydrogenase is an enzyme complex localized to the mitochondrial inner membrane and serves as one of the key hubs linking the respiratory electron transport chain to oxidative phosphorylation. Deficiency in SDH can lead to various disorders, including neurometabolic disturbances and tumor formation.

SDH catalyzes the dehydrogenation of succinate to fumarate; simultaneously, an electron transfer agent transfers electrons to 2,6-dichlorophenolindophenol (DCPIP), reducing the reduced form of DCPIP to its oxidized form. This reduced product exhibits a characteristic absorption peak at 600 nm; therefore, the enzymatic activity of SDH is calculated based on the decrease in absorbance at this wavelength.

When using this kit to analyze tissue and cell samples, it is necessary to determine the total protein concentration; the BCA method (Catalog No.: K001) is recommended.

Microplate reader (590–610 nm, optimal detection wavelength: 600 nm),

Normal saline (0.9% NaCl), or PBS (0.01 M, pH 7.4)

1.Before testing, all reagents should be allowed to equilibrate at room temperature.

2.Working solution preparation:

Mix Reagent 1, Reagent 4, Reagent 5, and Reagent 6 at a volume ratio of 14:2:2:1; prepare immediately before use; prepare only as needed; store in dark; use within the same day.

1.Sample Processing

Tissue sample preparation: Take 0.1 g of fresh tissue sample, rinse with 0.9% NaCl at 2–8 °C, blot dry with filter paper, weigh the sample, and place it into a homogenization container. Add 0.9 mL of Reagent 1 and 10 μL of Reagent 3, then perform mechanical homogenization; centrifuge at 4 °C, 600 × g for 5 min; collect the supernatant and discard the precipitate. Centrifuge again at 4 °C, 15,000 × g for 10 min; the resulting precipitate represents the extracted mitochondria. Add 200 μL of Reagent 2 and 2 μL of Reagent 3 to the precipitate, mix thoroughly, ultrasonicate for 5 min, and then centrifuge at 4 °C, 15,000 × g for 10 min. Collect the supernatant in an ice-cold container for subsequent analysis, while retaining a portion of the supernatant for protein concentration determination.

Cell sample: Take 2 × 10^6 collected cells and mix them with 400 μL Reagent 1 and 4 μL Reagent 3; homogenize the mixture, then centrifuge at 600 × g for 5 min at 4°C. Collect the supernatant and discard the precipitate. Next, centrifuge the mixture at 15,000 × g for 10 min at 4°C; the resulting precipitate represents the extracted mitochondria. Add 200 μL Reagent 2 and 2 μL Reagent 3 to the precipitate and mix well. Sonicate for 5 min, then centrifuge at 15,000 × g for 10 min at 4°C. Collect the supernatant and store it in an ice box for subsequent analysis; retain a portion of the supernatant for protein concentration determination.

2.Sample dilution

Prior to the formal assay, select 2–3 samples with expected significant differences, dilute them into different concentrations for a preliminary experiment; based on the results of this preliminary experiment and considering the linear range of this kit (0.83–65.42 U/L), perform the final dilution according to the table below (for reference only):

Note: The diluent is Reagent II.

①The supernatant of the homogenization sample should be analyzed on the same day.

② Store certain reagents away from light in accordance with the manufacturer's instructions.

③ It is recommended to limit the number of samples tested in a single experiment to no more than 8 samples.

Formula for calculating succinate dehydrogenase (SDH) activity in tissues and cells:

Definition: At room temperature, one enzyme activity unit is defined as the amount of SDH enzyme required to hydrolyze a substrate from every gram of tissue or cellular protein per minute, yielding 1 μmol of product.

[Note]

ΔATest: OD change of the test well(A1–A2)

ΔABlank: OD change of the blank well(A1–A2)

f: Dilution factor before adding the sample to the detection system

VTotal: Total volume of the reaction mixture (mL): 0.21 mL

VSample: Sample volume (mL), 0.02 mL

21.8*： molar absorptivity

Cpr: Sample protein concentration: gprot/L

T: Reaction time, 3 min

1000*：1 mmol/L=1000 μmol/L

1. This kit is intended solely for research purposes; should it be used for clinical diagnosis or any other purpose, our company shall not be liable for any issues arising therefrom nor shall we assume any legal responsibility.

2. Before conducting the experiment, please carefully read the instruction manual, properly adjust the instruments, and strictly follow the instructions when performing the experiment.

3. During the experiment, please wear lab coats and latex gloves to ensure proper protection.

4. The detection range of the kit does not correspond to the concentration range of the analyte in the sample. If the concentration of the analyte in the sample is too high or too low, please appropriately dilute or concentrate the sample.

5. If the sample being tested does not fall within the list of sample types specified in the instruction manual, it is recommended to first conduct a pilot experiment to verify its detection efficacy.

6. The final experimental results are closely related to factors such as the efficacy of the reagents, the operator's procedural skills, and the experimental environment. Our company assumes sole responsibility only for the kit itself and is not liable for any sample consumption resulting from its use; therefore, please carefully estimate the potential sample volume required before use and ensure sufficient sample reserves are available.