Recombinant Human VAP-1 Protein, CF

Catalog # Availability Size / Price Qty
3957-AO-010
Product Details
FAQs
Supplemental Products
Reviews

Recombinant Human VAP-1 Protein, CF Summary

Purity
>95%, 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 benzylamine. The specific activity is >8 pmol/min/μg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human VAP-1/AOC3 protein
Gly27-Asn763, with an N-terminal Met and 10-His tag, followed by a Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ile-Glu-Gly-Arg linker
Accession #
N-terminal Sequence
Analysis
Met
Structure / Form
Disulfide-linked homodimer
Predicted Molecular Mass
85 kDa
SDS-PAGE
80-90 kDa, reducing conditions

Product Datasheets

3957-AO

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: 50 mM HEPES, pH 7.5 
  • Recombinant Human VAP‑1/AOC3 (rhVAP‑1) (Catalog # 3957-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: Benzylamine (Sigma, Catalog # B5136), 100 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 rhVAP-1 to 20 ng/µL in Assay Buffer.
  2. Prepare the Substrate mixture 2 mM Benzylamine, 2 units/mL HRP and 100 µM AUR in Assay Buffer.
  3. In a plate, load 50 µL of 20 ng/µL rhVAP-1 and start the reaction by adding 50 µL of the Substrate mixture (step 2). Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of the Substrate mixture.
  4. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode for 5 minutes. Note: A cutoff must be set 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, 1 mM Benzylamine, and a curve of
     Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

Per Well:
  • rhVAP-1: 1.0 µg
  • Benzylamine: 1 mM
  • 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.

=
÷

Background: VAP-1/AOC3

Vascular adhesion protein-1 (VAP-1) is a copper amine oxidase with a topaquinone cofactor. VAP-1 is a Type II integral membrane protein, but a soluble form of the enzyme is present in human serum, and its level increases in diabetes and some inflammatory liver diseases (1, 2). VAP-1 catalyzes the oxidative deamination of small primary amines such as methylamine, benzylamine, and aminoacetone in a reaction that produces an aldehyde, ammonia, and H2O2 (3). The enzyme is sensitive to inhibition by semicarbazide. VAP-1 expression is highest in the endothelium of lung, heart, and intestine, but low in tissues such as brain, spleen, kidney, and liver (4). VAP-1 vascular expression is regulated at sites of inflammation through its release from intracellular granules in which the protein is stored (5). The adhesive function of VAP-1 has been demonstrated in studies showing that the protein is important for the adherence of certain lymphocyte subtypes to inflamed endothelial tissues (6). VAP-1 mediated adhesion is involved in the process of leukocyte extravasation, an important feature of inflammatory responses. The role of VAP-1 amine oxidase activity in this process is not fully defined, but it appears to be carbohydrate-dependent (7). VAP-1 is considered to be a therapeutic target for diabetes, oxidative stress, and inflammatory diseases (8).

References
  1. Kurkijärvi, R. et al. (1998) J. Immunol. 161:1549.
  2. Gearing, A.J.H. and W. Newman (1993) Immunol. Today 14:506.
  3. Lizcano, J.M. et al. (1998) Biochem. J. 331:69.
  4. Smith, D.J. et al. (1998) J. Exp. Med. 188:17.
  5. Jaakkala K. et al. (2000) Am. J. Pathol. 157:463.
  6. Salmi, M. and J. Jalkanen (2001) Trends Immunol. 22:211.
  7. Salmi, M. and J. Jalkanen (1996) J. Exp. Med. 183:569.
  8. Dunkel, P. et al. (2008) Curr. Med. Chem. 15:1827.
Long Name
Vascular Adhesion Protein-1
Entrez Gene IDs
8639 (Human); 11754 (Mouse); 29473 (Rat)
Alternate Names
amine oxidase, copper containing 3 (vascular adhesion protein 1); AOC3; Copper amine oxidase; EC 1.4.3; HPAO; HPAOSSAO; Semicarbazide-sensitive amine oxidase; SSAO; VAP1; VAP-1; VAP1EC 1.4.3.21; VAP-1membrane primary amine oxidase; Vascular adhesion protein 1

FAQs

No product specific FAQs exist for this product, however you may

View all Proteins and Enzyme FAQs

Reviews for Recombinant Human VAP-1 Protein, CF

There are currently no reviews for this product. Be the first to review Recombinant Human VAP-1 Protein, CF and earn rewards!

Have you used Recombinant Human VAP-1 Protein, 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