Alcohol dehydrogenase Activity Assay Kit (BA0011)

Product Name:	Alcohol dehydrogenase Activity Assay Kit (BA0011)
SKU:	BA0011
Detection Method:	Colorimetric kinetic (OD 565 nm)
Detection Range:	0.4 to 80 U/L (20 µL sample, 30 min); detection limit 0.1 U/L (120 min)
Sample Type:	['Plasma', 'Serum', 'Urine', 'Tissue', 'Culture media']
Species Reactivity:	All
Assay Time:	30 minutes (extendable to 2 hours)
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	-20°C
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

Alcohol dehydrogenase (ADH) is an oxidoreductase which catalyses the interconversion of alcohols and aldehydes or ketones. ADH is important in humans and other organisms for the breakdown of alcohols which may otherwise be toxic. In yeast and some bacteria, ADHs catalyse the opposite reaction and produce alcohol as part of fermentation. This non-radioactive, colorimetric ADH 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 increase in absorbance at 565 nm is directly proportional to the enzyme activity.

Fast and sensitive. Linear detection range (20 µL sample): 0.4 to 80 U/L for a 30 min reaction. Detection limit of 0.1 U/L for a 120 min reaction.
Convenient and high-throughput. Homogeneous mix-incubate-measure type assay that can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

ADH activity determination in biological samples (e.g. plasma, serum, urine, tissue and culture media).

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 50 mg in approximately 200 µL buffer containing 50 mM potassium phosphate (pH 7.5) and centrifuge at 10,000 x g for 15 min at 4°C, using 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 50 mM potassium phosphate buffer (pH 7.5) and centrifuge at 10,000 x g for 15 min at 4°C.
2	Equilibrate reagents to the desired reaction temperature (e.g. 25°C or 37°C) and briefly centrifuge tubes before use. Prepare the Working Reagent by mixing, for each 96-well assay, 5 µL Substrate, 14 µL MTT Solution, 9 µL NAD Solution, 1 µL Diaphorase and 55 µL Assay Buffer. Prepare the Blank Working Reagent by mixing 14 µL MTT Solution, 9 µL NAD Solution, 1 µL Diaphorase and 60 µL Assay Buffer (no Substrate); fresh reconstitution is recommended.
3	Transfer 100 µL H2O (ODH2O) and 100 µL Calibrator (ODCAL) solution into wells of a clear flat-bottom 96-well plate.
4	Transfer 20 µL sample into two separate wells, add 80 µL Working Reagent to one sample well and 80 µL Blank Working Reagent to the other, then tap the plate briefly to mix.
5	Read OD565nm (OD0), and again after 30 min (OD30) on a plate reader.

Subtract OD₀ from OD₃₀ for each sample and blank well to compute the change in OD for the sample and blank respectively. ADH Activity = [(change in OD_s - change in OD_b) / (OD_CAL - OD_H2O)] x (273 / t) x n (U/L), where t is the reaction time (30 min recommended) and n is the dilution factor. If sample ADH activity exceeds 80 U/L, use a shorter reaction time or dilute samples in water; for activity below 1 U/L the incubation can be extended to 2 hours. Unit definition: 1 Unit (U) of ADH will catalyse the conversion of 1 µmole of ethanol to acetaldehyde per min at pH 8.2.

Component	Quantity	Storage
Assay Buffer	10 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
Substrate (10% Ethanol)	1 mL	-20°C