Product Name:	LDH assay kit / Lactate Dehydrogenase Assay kit (Colorimetric) (BA0006)
SKU:	BA0006
Detection Method:	Colorimetric kinetic (OD 565 nm)
Detection Range:	2 to 200 IU/L; detection limit 2 IU/L
Sample Type:	['Serum', 'Plasma', 'Tissue', 'Cell lysate']
Species Reactivity:	All
Assay Time:	Within 30 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	-20°C
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

Lactate dehydrogenase (LDH) is an oxidoreductase that catalyses the interconversion of lactate and pyruvate. When disease or injury affects tissues containing LDH, the cells release LDH into the bloodstream, where it is identified at higher than normal levels; LDH is therefore most often measured to evaluate the presence of tissue or cell damage. This non-radioactive colorimetric LDH assay is based on the reduction of the tetrazolium salt MTT in an NADH-coupled enzymatic reaction to a reduced form of MTT which exhibits an absorption maximum at 565 nm. The intensity of the purple colour formed is directly proportional to the enzyme activity.

High sensitivity and wide linear range. Use 3 µL serum or plasma sample. The detection limit is 2 IU/L, linear up to 200 IU/L.
Homogeneous and simple procedure. Simple mix-and-measure procedure allows reliable quantitation of LDH activity within 30 minutes.
Robust and amenable to HTS. All reagents are compatible with high-throughput liquid handling instruments.

Direct Assays: LDH activity in serum, plasma and other sources.
Characterisation and Quality Control for LDH production.
Drug Discovery: screen and evaluation of LDH modulators.

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	Prepare samples: serum and plasma are assayed directly. For tissue, rinse in phosphate buffered saline (pH 7.4) to remove blood, homogenise in 5 mL buffer containing 100 mM potassium phosphate (pH 7.0) and 2 mM EDTA per gram tissue, centrifuge at 10,000 x g for 15 min at 4°C and use the supernatant. For cell lysate, collect cells by centrifugation at 2,000 x g for 5 min at 4°C, homogenise or sonicate in cold buffer containing 100 mM potassium phosphate (pH 7.0) and 2 mM EDTA, centrifuge at 10,000 x g for 15 min at 4°C and use the supernatant.
2	Equilibrate reagents to the desired reaction temperature (e.g. 25°C or 37°C) and briefly centrifuge reagent tubes before use. Prepare sufficient Working Reagent by mixing, for each well, 14 µL MTT Solution, 8 µL NAD Solution, 1 µL Diaphorase and 175 µL Substrate Buffer; fresh reconstitution is recommended.
3	Transfer 200 µL H2O (ODH2O) and 200 µL Calibrator (ODCAL) solution into separate wells of a clear flat-bottom 96-well plate.
4	Transfer 10 µL of each sample into separate wells and then add 190 µL Working Reagent to each sample well. Tap the plate briefly to mix.
5	Read OD565nm immediately (ODS0), and again after 25 min (ODS25) on a plate reader.

LDH Activity = [(OD_S25 - OD_S0) / (OD_CAL - OD_H2O)] x 43.68 x n (IU/L), where OD_S25 and OD_S0 are OD_565nm values of the sample at 25 min and 0 min, OD_CAL and OD_H2O are OD_565nm values of the Calibrator and water, and n is the dilution factor. If sample LDH activity exceeds 200 IU/L, dilute samples in water and repeat the assay. Unit definition: 1 Unit (IU) of LDH will catalyse the conversion of 1 µmole of lactate to pyruvate per min at pH 8.2.

Component	Quantity	Storage
Substrate Buffer	20 mL	-20°C
NAD Solution	1 mL	-20°C
MTT Solution	1.5 mL	-20°C
Diaphorase	120 µL	-20°C
Calibrator	1.5 mL	-20°C

LDH assay kit / Lactate Dehydrogenase Assay kit (Colorimetric) (BA0006)

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