D-Lactate Dehydrogenase (D-LDH) Activity Assay Kit (DNPH, Micro Method) - BioReagent, high purity

Cat. No.: D1505517

GRADE & PURITY BioReagent

Overview

Lactate dehydrogenase (LDH) is a glycolytic enzyme widely found in animals, plants, microorganisms, and cultured cells, with relatively high content in the kidneys. LDH is the terminal enzyme of the glycolytic pathway, catalyzing the reversible reaction between pyruvate and lactate, accompanied by the interconversion of NAD⁺/NADH. Based on the stereospecificity for the lactate substrate, LDH can be classified into D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) and L-lactate dehydrogenase (L-LDH, EC 1.1.1.27).

Detection Principle: D-LDH catalyzes the oxidation of D-lactate by NAD⁺ to generate pyruvate. Pyruvate further reacts with 2,4-dinitrophenylhydrazine to form pyruvate dinitrophenylhydrazone, which exhibits a brown-red color in an alkaline solution. The color intensity is proportional to the pyruvate concentration.

Detection Range: 0.031 - 2 μmol/mL
Sensitivity: 0.031 μmol/mL
Applicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma)

Component	48T	96T	Storage
Extraction Buffer	70 mL	70 mL×2	2-8℃
Reagent Ⅰ	7 mL	14 mL	2-8℃
Reagent Ⅱ	1EA	1EA	-20℃. Store in the dark.
Reagent Ⅲ	7 mL	14 mL	2-8℃
Reagent Ⅳ	20 mL	40 mL	2-8℃
Reagent Ⅴ	100 μL	200 μL	-20℃. Store in the dark.
Standard (100μmol/mL)	1 mL	1 mL	2-8℃. Store in the dark.

Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.  

User-Prepared Instruments and Reagents:

1.Microplate reader or visible spectrophotometer (capable of measuring absorbance at 450 nm)

2.96-well plate or micro glass cuvettes, adjustable micropipettes and tips

3.Constant temperature water bath, ice maker, centrifuge

4.Deionized water

5.Homogenizer (for tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent	Preparation	Notes
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 2-8°C
Reagent Ⅰ	Ready-to-use; equilibrate to room temperature before use.	Store at 2-8°C
Working Reagent Ⅱ	Prepare before use: For 48T, add 10 μL Reagent V to 1.3 mL deionized water. For 96T, add 20 μL Reagent V to 2.6 mL deionized water. Dissolve completely.	Unused reagent can be aliquoted and stored at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles.
Reagent Ⅲ	Ready-to-use; equilibrate to room temperature before use.	Store at 2-8°C
Reagent Ⅳ	Ready-to-use; equilibrate to room temperature before use.	Store at 2-8°C
Reagent Ⅴ	Ready-to-use; equilibrate to room temperature before use.	Store at -20°C protected from light
Standard	Ready-to-use; equilibrate to room temperature before use.	Store at 2-8°C protected from light

2. Standard Preparation

Using the 100 μmol/mL standard stock, prepare a dilution series as shown in the table below:

Tube	Standard Volume	Extraction Buffer Volume (μL)	Concentration (μmol/mL)
Std.1	20µL of 100μmol/mL	980	2
Std.2	100µL of Std.1	100	1
Std.3	100µL of Std.2	100	0.5
Std.4	100µL of Std.3	100	0.25
Std.5	100µL of Std.4	100	0.125
Std.6	100µL of Std.5	100	0.063
Std.7	100µL of Std.6	100	0.031
Blank	0	100	0

Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month. Control the thawing temperature and time during assay. If thawing at room temperature, complete within 4 hours.

3.1 Bacteria, Cells, or Tissue Samples

Bacteria or Cells: Collect bacteria or cells into a centrifuge tube, centrifuge, discard supernatant. Add Extraction Buffer at a ratio of bacteria/cell count (10⁴) to volume (mL) between 500:1 and 1000:1 (recommended: 5 million bacteria/cells in 1 mL Extraction Buffer). Sonicate in an ice bath for 5 min (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 min. Collect supernatant and keep on ice for assay.

Tissue: Add Extraction Buffer at a tissue mass (g) to volume (mL) ratio between 1:5 and 1:10 (recommended: weigh approx. 0.1 g tissue, add 1 mL Extraction Buffer). Homogenize in an ice bath. Centrifuge at 8,000 g, 4°C for 10 min. Collect supernatant and keep on ice for assay.

3.2 Serum (Plasma)
Assay directly.

4. Assay Steps

4.1 Preheat the microplate reader or visible spectrophotometer for 30 min. Set wavelength to 450 nm. For spectrophotometers, zero with deionized water.
4.2 Assay Procedure (perform in 1.5 mL microcentrifuge tubes):

Reagent	Test Tube (μL)	Control Tube (μL)	Standard Tube (μL)
Test Sample	10	10	0
Standard	0	0	10
Reagent Ⅰ	50	50	50
Working Reagent Ⅱ	10	0	0
Deionized Water	0	10	10

Mix thoroughly, incubate at 37°C for 15 min.

Reagent Ⅲ

50

50

50

Mix thoroughly, incubate at 37°C for 15 min.

Reagent Ⅳ

150

150

150

4.3 Mix thoroughly, let stand at room temperature for 30 min. Transfer 200 μL to a micro glass cuvette or 96-well plate. Measure absorbance at 450 nm, recorded as A_test, A_control, A_standard, A_blank. Calculate ΔA_test = A_test - A_control; ΔA_standard = A_standard - A_blank.

Note: The Blank and Standard Curve tubes need only be set up once. Each test sample requires a control tube. A preliminary test with 2-3 samples showing expected significant differences is recommended. If ΔA_test < 0.01, increase sample volume appropriately. If ΔA_test > 0.5, dilute sample further with Extraction Buffer (multiply result by dilution factor) or reduce sample amount used for extraction.

5. Calculation of Results

Note: We provide both derived and simplified calculation formulas. They are equivalent. The simplified formulas in bold are recommended for final calculation.

5.1 Standard Curve Plotting
Plot the standard concentration (x-axis) against ΔA_standard (y-axis) to generate the standard curve and obtain the standard equation. Substitute ΔA_test into the equation to obtain x (μmol/mL).

5.2 D-LDH Activity Calculation

(1) Based on Sample Volume
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of pyruvate per minute per mL of serum (plasma).
* Derived Formula: D-LDH Activity (U/mL) = x × V_sample ÷ V_sample ÷ T × 10³
* Simplified Formula: D-LDH Activity (U/mL) = 66.67 × x

(2) Based on Sample Mass
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of pyruvate per minute per gram of tissue.
* Derived Formula: D-LDH Activity (U/g mass) = x × V_sample ÷ (W ÷ V_{total sample} × V_sample) ÷ T × 10³
* Simplified Formula: D-LDH Activity (U/g mass) = 66.67 × x ÷ W

(3) Based on Bacterial or Cell Count
Unit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of pyruvate per minute per 10⁴ bacteria or cells.
* Derived Formula: D-LDH Activity (U/10⁴) = x × V_sample ÷ (N ÷ V_{total sample} × V_sample) ÷ T × 10³
* Simplified Formula: D-LDH Activity (U/10⁴) = 66.67 × x ÷ N

Parameter Definitions:

1.V_sample: Volume of sample added to the reaction system (0.01 mL)

2.V_{total sample}: Volume of Extraction Buffer added (1 mL)

3.T: Reaction time (15 min)

4.W: Sample mass (g)

5.N: Number of cells or bacteria (in units of 10⁴)

6.10³: Unit conversion factor (1 μmol/mL = 10³ nmol/mL)

Precautions

1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.

2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 6 months under corresponding storage conditions.