>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
<1.0 EU per 1 μg of the protein by the LAL method.
41 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Prepare 1X Assay Buffer by diluting 10X Assay Buffer in deionized water.
Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock.
Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of 1X Assay Buffer.
Prepare Substrate Mixture composed of 0.364 mM ATP and 3.64 mM Adenosine in 1X Assay Buffer.
Dilute rhADK to 50 ng/µL in 1X Assay Buffer.
Load 20 µL of the 50 ng/µL rhADK into the plate in triplicate. Include a control containing 20 µL of 1X Assay Buffer.
Add 55 µL of Substrate Mixture to the wells, excluding the standard curve and curve blank.
Incubate sealed at 37 °C for 1 hour.
Dilute Coupling Phosphatase 4 (supplied in kit) to 2.67 µg/mL in 1X Assay Buffer.
Add 100 µL of 1X Assay Buffer to each well containing the standard curve and curve blank.
Add 75 µL of 2.67 µg/mL Coupling Phosphatase 4 to wells containing enzyme and blanks, excluding the standard curve and curve blank.
Incubate sealed plate at room temperature for 5 minutes.
Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
Read plate at 620 nm (absorbance) in endpoint mode.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg)
*Derived from the phosphate standard curve using linear fitting and adjusted for Substrate Blank. **Use the sum of the incubation times prior to the addition of Malachite Green reagents due to the constant conditions throughout the assay.
Note: No coupling rate is applicable for this assay.
rhADK: 1.0 µg
Coupling Phosphatase 4: 0.2 µg
ATP: 20 nmol
Adenosine: 200 nmol
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Adenosine Kinase/ADK Protein, CF
Adenosine kinase (ADK) converts adenosine into 5'-adenosine-monophosphate. ADK is therefore key in regulating the concentration of adenosine, an essential homeostatic and metabolic regulator in all living systems (1). Adenosine not only activates specific signaling pathways by binding to adenosine receptors but also is a primordial metabolite and regulator of numerous biochemical reactions that are related to metabolism and genetics. ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. For example, inhibiting ADK activity promotes the replication of primary insulin-producing beta cells in diabetic mouse, rat, and pig (2); mutation of ADK causes hypermethioninemia, a condition with an excess of methionine in the blood (3); ADK is also identified as a neuropathological marker of the epileptic brain (4). Given its significant roles in various diseases, ADK is a rational therapeutic target (1). ADK is widely expressed in all tissues, with elevated levels in placenta, liver, muscle and kidney (5). The enzymatic activity of recombinant human ADK is measured using a phosphatase-coupled method (6).
Boison, D. (2013) Pharmacol. Rev. 65:906.
Annes, J.P. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:3915.
Bjursell, M.K. et al. (2011) Am. J. Hum. Genet. 89:507.
Aronica, E. et al. (2013) Neurochem. Int. in press.
Spychala, J. et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:1232.
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