Lactate dehydrogenase activity (WST-8 method) microcalorimetry instruction manual

Lactate dehydrogenase（LDH）Active Trace Method ManualPrinciple of MeasurementIt's through testingLDHDuring the process of catalyzing the reduction of pyruvic acid to lactic acidNADHTo indirectly determine the amount of generationLDHThe activity.

The specific process is as follows:

Reaction principle:inLDHUnder its influence,NAD+Generated by restorationNADH.NADHandWST-8The interaction generates color, which is then used for colorimetric measurement (λ)max=450 nm）.

Reaction conditions:LDHTranslate into EnglishNADRestore toNADH，NADHandWST-8Interaction produces color. This reaction takes place on ice in the dark, with each component (such as...)Assay BufferTheLactateTheNADTheWST-8TheEnhancer）It needs to be removed from the refrigerator and equilibrated to room temperature before the experiment, and the entire process should be carried out in the dark.

Absorption measurement:in450 nmMeasure absorbance at wavelength, absorbance andLDHThe activity is proportional and can be determined by colorimetric methodLDHThe activity.

Experimental steps and required equipment:

Experimental steps:Prepare working fluid（1×Assay BufferTheNADTheWST-8TheEnhancerandLactateMix evenly), add the working fluid96Add the sample to be tested into an orifice plate or micro glass colorimetric dish,37Measure the absorbance after incubating at a constant temperature of ℃ for a certain period of time.

Required equipment:ELISA reader or visible spectrophotometer (capable of measuring)450 nmThe absorbance of the sample, constant temperature box, ice maker, low-temperature centrifuge96Orifice plate or micro glass colorimetric dish, adjustable pipette and tip, deionized water, homogenizer (if it is a tissue sample).

Experimental precautions‌：

Reagent storage:Each component (such asAssay BufferTheLactateTheNADTheWST-8TheEnhancer）It needs to be stored on ice in the dark to avoid repeated freezing and thawing.

Precautions for operation:The entire experimental process needs to be conducted on ice in the dark to avoid light affecting the results.

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