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Recombinant Human ABP1/AOC1 Protein, CF

Catalog #: 8298-AO Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
8298-AO-010
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Recombinant Human ABP1/AOC1 Protein, CF Summary

Product Specifications

Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to produce hydrogen peroxide during the oxidation of histamine. The specific activity is >40 pmol/min/μg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human ABP1/AOC1 protein
Glu20-Val751, with a N-terminal 6-His tag
Accession #
CAA55046
N-terminal Sequence
Analysis
Inconclusive, His predicted. Protein identity confirmed by MS analysis of tryptic fragments.
Predicted Molecular Mass
84 kDa
SDS-PAGE
83-92 kDa, reducing conditions

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8298-AO

Product Datasheet
COA
SDS

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.

8298-AO

Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl, 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.
  • 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: 50 mM HEPES, pH 7.5
  • Recombinant Human ABP1/AOC1 (rhABP1) (Catalog # 8298-AO)
  • Coupling Enzyme:  Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  • Substrate Component 1: Histamine (Sigma, Catalog # H7250), 20 mM stock in deionized water
  • Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhABP1 to 1 ng/µL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 10 µM Histamine, 2 units/mL HRP, and 100 µM AUR in Assay Buffer.
  3. Load 50 µL of 1 ng/µL rhABP1 into wells of a plate and start the reactions by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing Assay Buffer instead of rhABP1.
  4. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode. Note:  A cutoff must be set manually at a wavelength of 570 nm.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 5 µM Histamine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

Per Well:
  • rhABP1: 0.05 µg
  • Histamine: 5 µM
  • HRP: 1 unit/mL
  • AUR: 50 µM
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.

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Background: ABP1/AOC1

Amiloride binding protein (ABP1), also known as diamine oxidase (DAO) and amine oxidase copper domain-containing protein 1 (AOC1), is an approximately 110 kDa member of the copper-containing amine oxidase family (1, 2). ABP1 is a glycosylated enzyme that forms disulfide-linked homodimers (3). It is stored in cytoplasmic granules of epithelial cells in the kidney, placenta, uterus, lung, and intestine (1, 4), and its extracellular release can be induced by heparin (5). ABP1 exhibits a substrate preference for histamine and putrescine, generating hydrogen peroxide and ammonia in an oxidative deamination reaction (3). ABP1 activity plays an important role in the catabolism of histamine and other bioactive polyamines (6, 7). Deficiencies in ABP1 can result in histaminosis or dietary histamine intolerance (8, 9).

References
  1. Novotny, W.F. et al. (1994) J. Biol. Chem. 269:9921.
  2. Barbry, P. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7347.
  3. McGrath, A.P. et al. (2009) Biochemistry 48:9810.
  4. Liang, X.H. et al. (2010) Endocrinology 151:5007.
  5. Klocker, J. et al. (2004) Vascul. Pharmacol. 40:293.
  6. Jones, B.L. and G.L. Kearns (2011) Clin. Pharmacol. Ther. 89:189.
  7. Bieganski, T. et al. (1983) Biochim. Biophys. Acta 756:196.
  8. Sattler, J. and W. Lorenz (1990) J. Neural Transm. Suppl. 32:291.
  9. Maintz, L. and N. Novak (2007) Am. J. Clin. Nutr. 85:1185.
Long Name
Amiloride Binding Protein 1
Entrez Gene IDs
26 (Human)
Alternate Names
ABP; ABP1; amiloride binding protein 1 (amine oxidase (copper-containing)); Amiloride-binding protein; amiloride-sensitive amine oxidase [copper-containing]; amiloride-sensitive amine oxidase; AOC1; AOC1DAOamiloride-binding protein-1; DAO1; Diamine Oxidase; EC 1.4.3; EC 1.4.3.22; Histaminase; KAO; Kidney amine oxidase

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