Source E. coli-derived, full length (1-348 aa) with an N-terminal His tag
Accession# P00330
Predicted Molecular Mass 38 kDa
SPECIFICATIONS
SDS PAGE 38 kDa, reducing conditions.
Activity
Specific activity: > 300 Units/mg One unit will convert 1.0 µmole of ethanol to acetaldehyde per minute at pH 9.2 at 25 °C.
Endotoxin Level < 1.0 EU per 1 μg of the protein by the LAL method.
Purity > 97%, by SDS-PAGE under reducing conditions and visualized by Coomassie Blue stain at 2 μg per lane.
Formulation Supplied as a 0.2 μm filtered solution in 25 mM Tris/HCl, pH 7.5; 100 mM NaCl;1 mM DTT.
PREPARATION AND STORAGE
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Avoid repeated freeze-thaw cycles. •6 months from date of receipt, -80 ◦C as supplied. •3 months, -80 ◦C under sterile conditions after opening.
ACTIVITY ASSAY PROTOCOL
Materials •Assay Buffer: 50 mM sodium pyrophosphate buffer. (pH 9.2) •Enzyme Diluent: 10 mM Sodium Phosphate Buffer, pH 7.5 with 0.1% (w/v) Bovine Serum Albumin •Recombinant Saccharomyces cerevisiae alcohol dehydrogenase, rsADH1 •Anhydrous ethanol •β-NAD+, 15 mM stock in deionized water •96-well Clear Plate (BNB, Catalog # 9G-DP33VR-N-LB) •Plate Reader (Model: Tecan Infinite M200 Pro) or equivalent •Enzyme Solution: Alcohol Dehydrogenase is unstable in solution and should be assayed immediately following preparation of solutions. Prepare a 1 mg/ml solution of Alcohol Dehydrogenase in cold (2–8 °C) 10 mM Sodium Phosphate Buffer, pH 7.5. Then dilute 0.02 ml of the 1 mg/ml solution to 10.0 ml with cold Enzyme Diluent.
Assay Procedure
1. Into appropriate EP tubes, accurately pipette the following:
2. Mix by inversion and equilibrate to 25 °C. Then add:
3. Immediately mix by inversion and pipette 250 μl to 96-well Clear Plate. Record the increase in A340 for ~6 minutes. Obtain the A340nm/minute using the one to six minute range for both the Test and Blank. 4.Calculate specific activity: Specific Activity (μmol/min/mg) =
*Adjusted for Substrate Blank **Using the extinction coefficient 6270 M-1cm-1 ***Using the path correction 0.8 cm
Final assay conditions Per Well: •rsADH1: 16.7 ng •NAD+: 7.5 mM •Alcohol: 3.3% (v/v)
DATA
BACKGROUND Alcohol dehydrogenase; fermentative isozyme active as homo- or heterotetramers; required for the reduction of acetaldehyde to ethanol, the last step in the glycolytic pathway.