Babco, Signet, Sternberger Monoclonals, Senetek, Covance

protein

Recombinant Human CD73 (carrier-free)

796106

100 μg

850 USD

BA

796106

Recombinant Human CD73 (carrier-free)

5'-nucleotidase ecto, Ecto-5'-Nucleotidase, 5'-Nucleotidase, Purine 5-Prime-Nucleotidase, NT, eN, NT5, NTE, eNT, CD73, E5NT

100 μg

850 USD

Human

ADD EXPRESSION SYSTEM HERE

RECOM

Human CD73 Activity Assay Recombinant human CD73 activity is measured by its ability to hydrolyze the 5'-phosphate group from the substrate adenosine-5'-monophosphate (AMP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit. The specific activity is > 10,000 pmol/min/μg, as measured in the presence of 0.001 μg of human CD73. Materials Assay buffer: 25 mM Tris, 5 mM MgCl2, pH 7.5;Human 5'-Nucleotidase/CD73 Substrate: Adenosine monophosphate (AMP) (Sigma, Cat. No. A1752), 5 mM stock in deionized water;Malachite Green Phosphate Detection Kit (Sigma, Cat. No. MAK307-1KT);96-well Clear Plate (Costar, Cat. No. 92592);Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalentActivity Assay Procedures 1. ;Prepare the dilution of human CD73 at 0.04 µg/mL in assay buffer.;2. ;In assay buffer, prepare a phosphate standard curve, first concentration should be 100 µM. The standard curve should contain a range of 0.039 to 2.5 nmol per well. 3. ;In a clear plate, load 25 µL of prepared human CD73 (0.04 µg/mL), standard curve, and blanks of assay buffer.;4. ;In assay buffer, dilute the substrate to 100 µM. Add 25 µl of substrate to all the wells. Ensure to mix well. 5. ;Parafilm the plate and incubate at 37ᵒC for 20 minutes. 6. ;Make a 1:100 dilution of Malachite Green Solution B in Solution A, respectively. For example, 10 µL Solution B should be mixed with 1 mL of Solution A. Mix well. 7. ;Add 10 µL of the mixture to each well. Mix well. At room temperature, incubate for 20 minutes. 8. ;Read at wavelength of 620 nm (absorbance) in endpoint mode. Calculate the specific activity using the following formula: Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/1 nmol)) (Incubation time (min) X amount of enzyme (µg)); • Adjusted for the substrate blank and derived from the phosphate standard curve. Final Assay conditions per well:;hCD73: 0.001 ug Substrate: 35.7 µM; BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are validated in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com;