Recombinant Human Methionine Aminopeptidase 1 Protein, CF

Catalog # Availability Size / Price Qty
3537-ZN-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Methionine Aminopeptidase 1 Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measure by its ability to remove methionine from a fluorogenic peptide substrate H-Met-Gly-Pro-AMC (Catalog # ES017). The resulting GP-AMC is cleaved by Recombinant Human DPPIV/CD26 (Catalog # 9168-SE). The specific activity is >200 pmol/min/µg, as measured under the described conditions. S
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Methionine Aminopeptidase 1/METAP1 protein
His52-Phe386 & Lys53-Phe386, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
His52 & Lys53
Predicted Molecular Mass
38 kDa
SDS-PAGE
38 kDa, reducing conditions

Product Datasheets

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3537-ZN

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.

3537-ZN

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Activation Buffer: 50 mM HEPES, 0.1 mM CoCl2, 0.1 M NaCl, pH 7.5
  • Assay Buffer: 25 mM Tris, pH 8.0
  • Recombinant Human Methionine Aminopeptidase 1/METAP1 (rhMETAP1) (Catalog # 3537-ZN)
  • Recombinant Human DPPIV/CD26 (rhCD26) (Catalog #  9168-SE)
  • Substrate Met-Gly-Pro-AMC (Catalog # ES017)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMETAP1 to 4 µg/mL in Activation Buffer.
  2. Dilute Substrate to 200 µM in Activation Buffer.
  3. Combine equal volumes of 4 µg/mL rhMETAP1 and 200 µM Substrate. Include a Substrate Blank containing Activation Buffer in place of rhMETAP1.
  4. Incubate reactions for 5 minutes at room temperature.
  5. Stop reactions by heating at 95-100 °C for 5 minutes.
  6. Cool reactions on ice for 3 minutes and centrifuge briefly.
  7. Dilute rhCD26 to 2 ng/µL in Assay Buffer.
  8. Load into a black well plate 50 µL of each incubated mixture and add 50 µL 2 ng/µL rhCD26 to each.
  9. Incubate at room temperature for 10 minutes.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
  11. Calculate specific activity using the following formula:

     Specific Activity (pmol/min/µg) =

Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)
Incubation time (min) x amount of enzyme (µg)

     *Adjusted for Substrate Blank

     **Derived using calibration standard 7-Amino-4-Methyl-Courmarin (AMC) (Sigma, Catalog # A-9891).

Per Well:
  • rhMETAP1: 0.10 µg
  • rhCD26: 0.10 µg
  • Substrate: 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: Methionine Aminopeptidase 1/METAP1

The human METAP1 gene encodes Methionine Aminopeptidase 1, a member of the M24 family of metalloproteases. METAPs catalyze the removal of the initiator methionine residue from nascent peptides (1) and are essential for cell growth (2). Inhibition of METAPs provides a novel strategy in developing anti-cancer drugs (3). The purified rhMETAP1 consists of amino acid residues 52/53 to 386. A previous report showed that the N-terminal 89 amino acid region was not essential for catalytic activity (4).

References
  1. Lowther, W.T. and B.W. Matthews (2000) Biochim. Biophys. Acta. 1477:157.
  2. Li, X. and Y.H. Chang (1995) Proc. Natl. Acad. Sci. USA 92:12357.
  3. Liu, S. et al. (1998) Science 282:1324.
  4. Addlagatta, A. et al. (2005) Biochemistry 44:14741.
Entrez Gene IDs
23173 (Human); 75624 (Mouse)
Alternate Names
DKFZp781C0419; EC 3.4.11.18; KIAA0094MAP 1; MAP1; MAP1A; MetAP 1; MetAP1; MetAP1A; Methionine Aminopeptidase 1; methionyl aminopeptidase 1; Peptidase M 1

FAQs

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Fluorogenic Peptide Substrates

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