Recombinant Human alpha-Aminoadipate Aminotransferase, CF

Catalog # Availability Size / Price Qty
7927-AT-010
R&D Systems Recombinant Proteins and Enzymes
1 Image
Product Details
FAQs
Reviews

Recombinant Human alpha-Aminoadipate Aminotransferase, CF Summary

Product Specifications

Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to form Kynurenic Acid from Kynurenine and alpha-Ketoglutarate. The specific activity is >170 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human alpha-Aminoadipate Aminotransferase protein
Met1-Leu425, with an N-terminal Gly-Arg-Ala-His
Accession #
N-terminal Sequence
Analysis
Gly
Predicted Molecular Mass
48 kDa
SDS-PAGE
40-42 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • 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
  1. Dilute 50 mM Kynurenic acid standard to 400 µM in Assay Buffer. This is the first point of the standard curve.
  2. Perform six additional one-half serial dilutions in Assay Buffer. The standard curve has a range of 156‑10,000 pmol per well.
  3. Dilute rhAADAT to 5 µg/mL in Assay Buffer.
  4. Prepare Substrate Component Solution containing 50 mM HEPES, 20 mM alpha -ketoglutarate, 80 µM Pyridoxal 5’-phosphate, pH 8.0.
  5. Immediately before use, prepare Substrate Mixture by combining 25 µL of Kynurenine with 50 µL of Substrate Component Solution, per well assayed.
  6. Load 25 µL of each standard curve dilution into empty wells of a black well plate.
  7. 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.
  8. Add 75 µL of Substrate Mixture to all wells used.
  9. Cover the plate with a plate sealer and incubate at room temperature for 30 minutes.
  10. Add 100 µL of KYNA Development Mixture to all wells used.
  11. Read plate at excitation and emission wavelengths of 338 and 405 nm, respectively, in endpoint mode.
  12. 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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

=
÷

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).

References
  1. Han, Q. et al. (2008) Biosci. Rep. 28:205.
  2. Passera, E. et al. (2011) FEBS J. 278:1882.
  3. Vecsei, L. et al. (2013) Nat. Rev. Drug Discov. 12:64.
  4. Wang, J. et al. (2006) J. Biol. Chem. 281:22021.
  5. Rossi, F. et al. (2008) J. Biol. Chem. 283:3559.
Long Name
alpha-Aminoadipate Aminotransferase
Entrez Gene IDs
51166 (Human); 23923 (Mouse); 29416 (Rat)
Alternate Names
AADAT; alphaAminoadipate Aminotransferase; alpha-Aminoadipate Aminotransferase; aminoadipate aminotransferase; EC 2.6.1.39,2-aminoadipate aminotransferase; EC 2.6.1.7,2-aminoadipate transaminase; KAT/AadAT; KAT2; KAT2mitochondrial; Kynurenine aminotransferase II; Kynurenine--oxoglutarate aminotransferase II; Kynurenine--oxoglutarate transaminase II; L kynurenine/alpha aminoadipate aminotransferase

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 FAQs

Reviews 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/¥1250 Yen for a review with an image

$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image

Submit a Review