One unit of enzyme catalyzes the reduction of 1 µmole NAD+ per minute at 25ºC and pH 8.8.
The activity of 0240-0504 is determined using a procedure as published by Vallee, B.L. and Hoch, F.L., Proc. Nat. Acad. Sci. USA, 41: 327-338 1955.
Application
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visitwww.abdserotec.com/protocols.
Application Name
Yes
No
Not Determined
Suggested Dilution
ELISA
Functional Assays
Western Blotting
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using the appropriate negative/positive controls.
Widely used for the determination of ethanol and as part of coupled-enzymic systems for the determination of metabolites in biological fluids.
Storage
Prior to reconstitution store at +4oC.
After reconstitution store at -20oC.
Storage in frost-free freezers is not recommended. This product should be stored undiluted.
Avoid repeated freezing and thawing as this may denature the antibody.
Should this product contain a precipitate we recommend microcentrifugation before use.
Reagents:
0.05 M Sodium pyrophosphate buffer, pH 8.8.
96% Ethanol (substrate).
0.025 M NAD+ (16.7 mg/ml) in buffer. Prepare fresh.
0.01 M Sodium phosphate buffer, pH 7.5, containing 0.1% bovine serum albumin (BSA).
Alcohol dehydrogenase (enzyme) - Dissolve sufficient amount of enzyme in 0.01 M sodium phosphate buffer containing 0.1% BSA, pH 7.5, to give a concentration of 0.1-0.5 U/ml. Prepare fresh immediately prior to assay.
Procedure:
1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25°C.
2. In a cuvette pipette the following reagents in the amounts indicated:
Sodium pyrophosphate buffer
1.4 ml
NAD+
1.4 ml
Ethanol (substrate)
0.1 ml
3. Incubate cuvette in spectrophotometer, at 25°C for 5 min. to achieve temperature equilibration and then record absorbance at 340 nm (blank).
4. Initiate the reaction by adding 0.1 ml of ADH (enzyme) solution to the cuvette. Record the increase in absorbance at 340 nm for 5 min.
5. Calculate the E340nm/min
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