Recombinant Human Methionine Aminopeptidase 1 Protein, CF
Recombinant Human Methionine Aminopeptidase 1 Protein, CF Summary
Product Specifications
His52-Phe386 & Lys53-Phe386, with a C-terminal 10-His tag
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.
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.
|
Assay Procedure
- 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
- Dilute rhMETAP1 to 4 µg/mL in Activation Buffer.
- Dilute Substrate to 200 µM in Activation Buffer.
- Combine equal volumes of 4 µg/mL rhMETAP1 and 200 µM Substrate. Include a Substrate Blank containing Activation Buffer in place of rhMETAP1.
- Incubate reactions for 5 minutes at room temperature.
- Stop reactions by heating at 95-100 °C for 5 minutes.
- Cool reactions on ice for 3 minutes and centrifuge briefly.
- Dilute rhCD26 to 2 ng/µL in Assay Buffer.
- Load into a black well plate 50 µL of each incubated mixture and add 50 µL 2 ng/µL rhCD26 to each.
- Incubate at room temperature for 10 minutes.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
- 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
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).
- Lowther, W.T. and B.W. Matthews (2000) Biochim. Biophys. Acta. 1477:157.
- Li, X. and Y.H. Chang (1995) Proc. Natl. Acad. Sci. USA 92:12357.
- Liu, S. et al. (1998) Science 282:1324.
- Addlagatta, A. et al. (2005) Biochemistry 44:14741.
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