| Usage | Self-supplied consumables:
Microplate reader or visible spectrophotometer (can measure the absorbance at 450 nm)
96-well plate or microglass cuvette, adjustable pipet-gun and gun head
thermostatic water bath, ice machine, centrifuge
deionized water
homogenizer Reagent preparation:
Extraction Buffer: read-to-use type; Before use, equilibrate to room temperature; Store at 4 ° C.
Reagent I: ready-to-use; Before use, equilibrate to room temperature; Store at 4 ° C.
Reagent II: prepared before use; Add 10&mu at 48 T; L Reagent V and 1.3 mL deionized water, 96 T added 20μ L Reagent V and 2.6 mL deionized water were fully dissolved until use. The reagents that were not used up were stored at -20 ° C after being packaged, and repeated freezing and thawing were prohibited.
Reagent III: ready to use; Before use, equilibrate to room temperature; Store at 4 ° C.
Reagent IV: ready-to-use; Before use, equilibrate to room temperature; Store at 4 ° C.
Reagent V: ready-to-use; Before use, equilibrate to room temperature; Store at -20 ° C.
Standard: ready-to-use; Before use, balance to room temperature; Store at 4 ° C.
standard preparation: use 100μ mol/mL standard, as shown in the table below, further dilute the standard: | NO. | Standard volume | Extraction Buffer volume（µL） | Concentration（μmol/mL） | | Std.1 | 20 µL 100 μmol/mL | 980 | 2 | | Std.2 | 100 µL of Std.1 (2 μmol/mL) | 100 | 1 | | Std.3 | 100 µL of Std.2 (1 μmol/mL) | 100 | 0.5 | | Std.4 | 100 µL of Std.3 (0.5 μmol/mL) | 100 | 0.25 | | Std.5 | 100 µL of Std.4 (0.25 μmol/mL) | 100 | 0.125 | | Std.6 | 100 µL of Std.5 (0.125 μmol/mL) | 100 | 0.063 | | Std.7 | 100 µL of Std.6 (0.063 μmol/mL) | 100 | 0.031 | | Blank | 0 | 100 | 0 |
Sample preparation:
Note: it is recommended to use fresh samples, if not immediately to experiment, the sample can store well for weeks in - 80 ℃. When measuring, the temperature and time of thawing should be controlled. When thawing at room temperature, the sample should be thawed within 4 hours.
1. Preparation of bacteria, cells or tissue samples: bacteria or cells: first collect bacteria or cells into a centrifuge tube, then discard the supernatant after centrifugation; According to the bacteria or cells (4) : Extraction Buffer volume (mL) is 500 ~ 1000: The ratio of 1 (recommended 5 million bacteria or cells added to 1 mL Extraction Buffer) was added to the Extraction Buffer, and the cells or bacteria were broken by ultrasound in an ice bath for 5 min (power 20% or 200 W, ultrasonic 3 s, interval 7 s, After centrifugation at 8000 g for 10 min at 4℃, the supernatant was removed and placed on ice for determination. Organization: according to the quality organization (g) : Extraction Buffer volume (mL) is the ratio of 1:5 ~ 10 (suggested take about 0.1 g group, add 1 mL Extraction Buffer) to Extraction Buffer and ice bath homogenate. 8000 g, 4 ℃ centrifuge for 10 min, take supernatant, the ice under test.
2, serum (plasma) : direct detection.
The experimental steps:
1, enzyme standard instrument or visible spectrophotometer preheating 30 min, adjust the wavelength of 450 nm. The visible spectrophotometer was zeroed with deionized water.
2, operation table (the following operation in 1.5 mL EP tube) :
| Reagent | Detection tube（μL） | Control tube（μL） | Standard tube（μL） | Blank tube（μL） | | Sample to be tested | 10 | 10 | 0 | 0 | | Standard | 0 | 0 | 10 | 0 | | Reagent I | 50 | 50 | 50 | 50 | | Reagent II | 10 | 0 | 0 | 0 | | Deionized water | 0 | 10 | 10 | 20 | | Fully blending, 37 ℃ water bath for 15 min | | Reagent III | 50 | 50 | 50 | 50 | | Fully blending, 37 ℃ water bath for 15 min | | Reagent IV | 150 | 150 | 150 | 150 |
3. Mix well and let stand for 30 min at room temperature, then transfer 200 μL to A microglass cuvette or 96-well plate to measure the absorbance at 450 nm, recorded as A assay, A control, A standard, and A blank, respectively. Determination of computing Δ A = A - A contrast, Δ A = A standard - A blank.
Note: Blank Wells and standard curves only need to be measured once, and a control well should be set up for each test well. Experiments suggest choose 2-3 expected before big differences of samples for preliminary experiments, if Δ A measurement is less than 0.01, can be appropriately increased sample size; If Δ A measuring more than 0.5, samples available Extraction Buffer further dilution, the calculation results multiplied by the dilution ratio, or reduce the Extraction with sample size.
The results were calculated as follows:
Note: We provide you with the calculation formula, including the derivation process calculation formula and the concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.
1. Drawing of the standard curve
The standard equation y=kx+b was obtained by drawing the standard curve with the concentration of the standard solution on the X-axis and the ΔA standard on the Y-axis. The ΔA determination was put into the equation to obtain x (μmol/mL).
2, D-LDH activity calculation
(1) calculated by sample volume
Definition of unit: per minute per mL serum (plasma) to make 1 nmol pyruvate is defined as a unit of enzyme activity.
D - LDH activity (U/mL) = x x V kind present present T V sample 10 3 = 66.67 x x x
(2) according to the sample quality is calculated
Definition of unit: 1 nmol pyruvate per minute catalyzed production per g of tissue was defined as one unit of enzyme activity.
Quality of D - LDH activity (U/g) = x x V sample present sample (W present V total sample (V) present T * 3 = 66.67 x 10 x present W
(3) According to the number of bacteria or cells
Definition of unit: every 10 four bacteria or cells per minute catalytic produce 1 nmol pyruvate is defined as a unit of enzyme activity.
D - LDH activity (U / 10 4) = x x V sample present sample (N present V total sample (V) present T * 3 = 66.67 x 10 x present N
V-like: the sample volume added to the reaction system, 0.01 mL; V sample total: volume of Extraction Buffer added, 1 mL; T: the reaction time, 15 min; W: sample quality, g; N: the number of cells or bacteria, tens of; 3:10 unit conversion factor, 1 mu mol/mL = 10 3 nmol/mL. |
| Theory | Lactate dehydrogenase (LDH) is a glycolytic enzyme that widely exists in animals, plants, microorganisms and cultured cells, among which the kidney has a high content. LDH is a terminal enzyme in the glycolytic pathway, which catalyzes the reversible reaction between pyruvate and lactate, accompanied by the interconversion between NAD+/NADH. According to the conformation of its catalytic substrate - lactate, it can be divided into D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) and L-lactate dehydrogenase (L-LDH, EC 1.1.1.27). D-LDH catalyzes NAD+ to oxidize D-lactic acid to pyruvate, which further interacts with 2, 4-dinitrophenylhydrazine to form dinitrophenylhydrazone pyruvate. In alkaline solution, the color of D-LDH is brownish red, and the color is proportional to the concentration of pyruvate. |
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