Recombinant Human alpha-Aminoadipate Aminotransferase, CF
Recombinant Human alpha-Aminoadipate Aminotransferase, CF Summary
Product Specifications
Met1-Leu425, with an N-terminal Gly-Arg-Ala-His
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.
7927-AT
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES and NaCl. |
| 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: 20 mM HEPES, 50 mM NaCl, pH 8.0
- Recombinant Human alpha ‑Aminoadipate Aminotransferase (rhAADAT) (Catalog # 7927-AT)
- L-Kynurenine (Sigma, Catalog # K8625), 40 mM stock in 80 mM HEPES, 8 mM HCl, pH 8.0
- alpha -Ketoglutarate (Sigma, Catalog # K2010), 1 M stock in diH2O
- Pyridoxal 5’-phosphate (Sigma, Catalog # P9255), 100 mM stock in 1 M HEPES, pH 8.0
- KYNA Development Mixture: 2 M ZnCl2, 2 M NaOAc in diH2O
- Standard: Kynurenic acid (Catalog # 0223), 50 mM stock in 100 mM NaOH
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
- Dilute 50 mM Kynurenic acid standard to 400 µM in Assay Buffer. This is the first point of the standard curve.
- Perform six additional one-half serial dilutions in Assay Buffer. The standard curve has a range of 156‑10,000 pmol per well.
- Dilute rhAADAT to 5 µg/mL in Assay Buffer.
- Prepare Substrate Component Solution containing 50 mM HEPES, 20 mM alpha -ketoglutarate, 80 µM Pyridoxal 5’-phosphate, pH 8.0.
- Immediately before use, prepare Substrate Mixture by combining 25 µL of Kynurenine with 50 µL of Substrate Component Solution, per well assayed.
- Load 25 µL of each standard curve dilution into empty wells of a black well plate.
- Load 25 µL of dilute rhAADAT into empty wells. Include a Blank containing 25 µL of Assay Buffer. This will serve as both enzyme and standard curve blank.
- Add 75 µL of Substrate Mixture to all wells used.
- Cover the plate with a plate sealer and incubate at room temperature for 30 minutes.
- Add 100 µL of KYNA Development Mixture to all wells used.
- Read plate at excitation and emission wavelengths of 338 and 405 nm, respectively, in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Kynurenic acid produced* (pmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the Kynurenic acid curve using linear fitting and adjusted for Blank.
Per Reaction:- rhAADAT: 0.125 µg
- alpha -ketoglutarate: 10 mM
- Pyridoxal 5’-phosphate: 40 µM
- Kynurenine: 10 mM
Background: alpha-Aminoadipate Aminotransferase
Kynurenine aminotransferase II (KAT‑II), also known as alpha ‑Aminoadipate Aminotransferase (AADAT), catalyzes the PLP‑dependent transamination of aminoadipate to alpha‑oxoadipate in the catabolism of lysine in the liver and also is the primary brain enzyme catalyzing the transamination of kynurenine to kynurenic acid (KYNA) (1). KYNA is an endogenous antagonist of the N‑methyl‑D‑aspartate (NMDA) receptors with weaker effects on kainite and alpha‑amino‑3‑hydroxy‑5-methyl‑4‑isoxazole (AMPA) receptors, the other two ionotropic glutamate receptors (2, 3). KYNA also acts upon alpha‑7 nicotinic acetylcholine receptors ( alpha 7 nAChRs) and importantly may suppress the pre-synaptic release of glutamate to confer neuroprotective effects against NMDA‑receptor mediated over‑stimulation. Also, KYNA is an endogenous ligand of the orphaned G protein-coupled receptor 35 (GPR35), found primarily in immune cells, and may induce inositol phosphate production and Ca2+ mobilizaiton (4). Elevated levels of KYNA have been implicated in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, schizophrenia and cognitive impairment (3, 5).
- Han, Q. et al. (2008) Biosci. Rep. 28:205.
- Passera, E. et al. (2011) FEBS J. 278:1882.
- Vecsei, L. et al. (2013) Nat. Rev. Drug Discov. 12:64.
- Wang, J. et al. (2006) J. Biol. Chem. 281:22021.
- Rossi, F. et al. (2008) J. Biol. Chem. 283:3559.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Human alpha-Aminoadipate Aminotransferase, CF
There are currently no reviews for this product. Be the first to review Recombinant Human alpha-Aminoadipate Aminotransferase, CF and earn rewards!
Have you used Recombinant Human alpha-Aminoadipate Aminotransferase, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
