Recombinant Human Adenosine Deaminase/ADA Protein, CF
Recombinant Human Adenosine Deaminase/ADA Protein, CF Summary
Product Specifications
Met1-Leu363, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
7048-AD
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM HEPES, pH 7.5
- Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
- Substrate: Adenosine (Sigma, Catalog # A9251), 10 mM stock in deionized water
- UV plate (Costar, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Thaw the Substrate stock and then warm it at 37 °C for 20 minutes. Mix well after incubation.
- Dilute rhADA to 0.1 ng/μL in Assay Buffer.
- Dilute Substrate to 200 μM in Assay Buffer.
- Load into a plate 50 μL of 0.1 ng/μL rhADA and start the reaction by adding 50 μL of 200 μM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
- Read plate at a wavelength of 265 nm (bottom read) in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x -1 x well volume (L) x 1012 pmol/mol |
| ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 8500 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhADA: 0.005 μg
- Substrate: 100 μM
Reconstitution Calculator
Background: Adenosine Deaminase/ADA
Adenosine Deaminase (ADA, adenosine aminohydrolase) is one of the key enzymes of purine nucleotide catabolism. It catalyses the hydrolytic deamination of adenosine and deoxy‑adenosine to inosine and deoxyinosine (1, 2). ADA is expressed in virtually all tissues and is expressed at high levels in T-lymphocytes. Adenosine Deaminase deficiency can cause a form of SCID (severe combined immunodeficiency) and lymphopenia in both B- and T-cell lineages (3, 4). ADA can be used as a sensitive diagnostic marker for tuberculous pleuritis (5). Although it is primarily a cytosolic enzyme, ADA is known to be a positive regulator of T-cell co‑activation due to its binding to CD26 at the cell surface. The interaction of ADA with CD26 regulates lymphocyte-epithelial cell adhesion (6).
- Wolfenden, R.V. et al. (1969) Biochemistry 6:2412.
- Lowenstein, J.M. (1972) Physiol. Rev. 52:382.
- Giblett, E.R. et al. (1972) Lancet 2:1067.
- Coleman, M.S. et al. (1978) J. Biol. Chem. 253:1619.
- Baba, K. et al. (2008) PLoS ONE 3:e2788.
- Gines, S. et al. (2002) Biochem. J. 361:203.
Citation for Recombinant Human Adenosine Deaminase/ADA Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Autocrine Adenosine regulates tumor polyfunctional CD73+CD4+ effector T cells devoid of immune checkpoints
Authors: N Gourdin, M Bossennec, C Rodriguez, S Vigano, C Machon, C Jandus, D Bauché, J Faget, I Durand, N Chopin, O Tredan, JC Marie, B Dubois, J Guitton, P Romero, C Caux, C Ménétrier-
Cancer Res., 2018-03-20;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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