Lactate dehydrogenase (LDH) Activity Test Kit (Micromethod)

Catalog Number: abs580236 Brand: Absin

Price:

$150.00

Size: 96T

Quantity:

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Product Details

Product Specification

Operating Instructions

Animal and plant tissues, cells, serum (plasma) and other liquid samples

Usage

Self-supplied consumables:
Microplate reader or visible spectrophotometer (can measure the absorbance at 450 nm)
thermostat, ice machine, low temperature centrifuge
96-well plate or microglass cuvette, adjustable pipet gun and tip
deionized water
/>Homogenizer (if it is a tissue sample)
Reagent preparation: Note: Before opening the cap of each component (tubule reagent), please centrifuge at low speed.
1× Assay Buffer: before use, Assay Buffer (5×) Dilute 5 times with deionized water to a concentration of 1× Assay Buffer, equilibrated to room temperature; Store at 4 ° C.
Lactate: ready-to-use; During the whole experiment, the ice was placed away from light; Aliquots were stored at -20 ° C in the dark.
NAD: ready-to-use; During the whole experiment, the ice was placed in the dark; Aliquots were stored at -20 ° C in the dark.
WST-8: ready-to-use type; During the whole experiment, the ice was placed away from light; Aliquots were stored at -20 ° C in the dark.
Enhancer: ready-to-use; Place on ice in the dark during the whole experiment; Aliquots were stored at -20 ° C in the dark.
Working Reagent: prepare 55 &micro per well; L working solution, ready for use: absorb 31 µ L 1× Assay Buffer, 8 µ L NAD, 5 µ L WST-8, 1 µ L Enhancer & 10 μ L Lactate mix well.
Lactate Dehydrogenase Standard (20 U/mL): put 200µ L Lactate Dehydrogenase Standard with 800μ L 1× Assay Buffer was diluted to 20U/mL and mixed well; Place on ice in the dark; Store in aliquots at -20 ° C.
standard curve Settings: as indicated in the table below, with 1× Assay Buffer was used to dilute the 20 U/mL standard into 20, 16, 12, 8, 4, 2, and 1 U/mL standard solutions.

NO.	20 U/mL Standard volume（µL）	1×Assay Buffer volume（µL）	Standard concentration（U/mL）
Std.1	200	0	20
Std.2	160	40	16
Std.3	120	80	12
Std.4	80	120	8
Std.5	40	160	4
Std.6	20	180	2
Std.7	10	190	1

Sample preparation:
Note: it is recommended to use fresh samples. If you don't use immediately, can be in the sample - 80 ℃ to save for a month.
1. Animal and plant tissues: weigh about 0.1 g of sample, add 1 mL of precooled 1×Assay Buffer, homogenize in an ice bath, centrifuge at 10,000 g at 4℃ for 15 min, take the supernatant and put it on ice for testing.
2, cells: Five million cells were collected into a centrifuge tube and washed with cold PBS. After centrifugation, the supernatant was discarded, and 1 mL of 1×Assay Buffer was added. The cells were sonicated in an ice bath for 5 min (20% power or 200 W, 3 s, 7 s interval, repeated 30 times). Then the samples were centrifuged at 10,000 g for 15 min at 4 ° C, and the supernatant was removed and placed on ice until measured.
3, serum, plasma and other liquid samples: direct determination.
Note: Protein quantification kit abs9232 (BCA assay) is recommended for protein concentration determination.

The experimental steps:
1. The microplate reader or visible spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 450 nm. The visible spectrophotometer was zeroed with deionized water.
2. Operation table (the following operations are operated in 96-well plate or microglass cuvette) :

Reagent	Detector tube（μL）	Standard tube（μL）	Blank tube（μL）
Sample on clear	0	0	50
Standard	0	50	0
Deionized water	50	0	0
Working Reagent	50	50	50

3, after mixing, 37 ℃ avoid light incubation for 30 min, measuring 450 nm absorbance, the blank hole for A short, standard hole as A standard, the determination of hole for A test. It was calculated that ΔA measured =A measured -A null, ΔA standard =A marked -A null.

Note: Before the experiment, it is recommended to select 2-3 samples with large expected differences for pre-experiment. If ΔA was less than 0.001, the sample size could be increased appropriately. If ΔA is greater than 2.0, the sample can be further diluted with 1×Assay Buffer, the calculated result multiplied by the dilution, or the sample size for extraction can be reduced.

The results were calculated as follows:
Note: we provide you with the formula, including the formula derivation and concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.
1, the drawing standard curve
The standard curve was plotted using the concentration of the standard solution as the Y-axis and ΔA as the X-axis.
2. Calculation of LDH content
The ΔA of the sample was substituted into the equation to obtain the y value (U/mL).
(1) Calculated according to fresh weight of sample
LDH content (U/g fresh weight)=y×V sample ÷(W×V sample ÷ total V sample)×n=y÷W×n
(2) calculated according to protein concentration
LDH content (U/mg protein)=y× V-like ÷(V-like ×Cpr)×n=y÷Cpr×n
(3) calculated by sample volume
LDH content (U/mL)=y×V sample ÷V sample ×n=y×n
(4) calculated by cell number
LDH content (U / 10 4 cells) = x y V sample present sample (500 x V present V sample total) (n = 500 x n y members present
V: join the sample volume, 0.05 mL; W: sample quality, g; V sample total: Add 1×Assay Buffer volume, 1 mL; n: sample dilution; Cpr: protein sample concentration, mg/mL; 500: total number of cells, 5 million.

Theory

Lactate dehydrogenase (LDH) is an oxidoreductase (EC1.1.1.27) that is found in a wide variety of organisms. It catalyzes the interconversion of pyruvate and lactate, which is accompanied by the interconversion of NADH and NAD+. Under hypoxic conditions, it converts pyruvate, the end product of glycolysis, to lactate. LDH quantification is clinically relevant because serum levels of certain LDH isoenzymes reflect pathological conditions in specific tissues. When disease, injury, or toxic substances damage tissues, the lactate dehydrogenase of the cells is released into the blood. Since lactate dehydrogenase is a fairly stable enzyme, it has been widely used to assess tissue and cell damage and toxicity. The CheKine™ Lactate dehydrogenase (LDH) Activity Assay Kit (microassay) provides a simple and convenient microassay for the determination of lactate dehydrogenase in animal and plant tissues, cells, serum (plasma), and other biological fluids. In this trace method, LDH NAD reduction as NADH, NADH and WST - 8 interact to produce color, thus carries on the colorimetric determination of lambda (Max = 450 nm).

Synonym

Micro Lactate Dehydrogenase (LDH) Assay Kit

Composition

Name	Size（96T）	Storage
Assay Buffer (5×)	25mL	4℃
Lactate	1.2mL	-20℃,keep in dark place
NAD	1mL	-20℃,keep in dark place
WST-8	600μL	-20℃,keep in dark place
Enhancer	120μL	-20℃,keep in dark place
Lactate Dehydrogenase Standard (100 U/mL)	0.5mL	-20℃,keep in dark place

Background

Lactate dehydrogenase is an oxidoreductase found in a variety of organisms. It catalyzes the interconversion of pyruvate and lactate, accompanied by the interconversion of NADH and NAD+. It converts pyruvate, the end product of glycolysis, to lactate under hypoxic conditions. Lactate dehydrogenase quantitative clinical significance, because some of serum lactate dehydrogenase isozyme level reflects the pathological condition of a particular organization. When disease, injury, or toxic agents damage tissues, cellular lactate dehydrogenase is released into the bloodstream. Because it is a fairly stable enzyme, lactate dehydrogenase is widely used to assess tissue and cell damage and toxicity.

General Notes

1. Before opening the lid, centrifugation was performed.
2, pre-test to ensure that the reading is within the standard range.
3, use a fresh sample. If not analyzed immediately, samples can be taken at -80° C store for one month.

Storage Temp.

-20℃, stored in the dark, valid for 6 months.

Test Range

1-20 U/mL

Applications

Animal and plant tissues, cells, serum (plasma) and other liquid samples

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Lactate dehydrogenase (LDH) Activity Test Kit (Micromethod)