Recombinant Human Methionine Aminopeptidase 1D Protein, CF
Recombinant Human Methionine Aminopeptidase 1D Protein, CF Summary
Product Specifications
Arg44-Ala335, with an N-terminal Met and 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.
4714-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and 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.
|
Assay Procedure
- Activation Buffer: 50 mM HEPES, 0.1 mM CoCl2, 100 mM NaCl, pH 7.5
- Assay Buffer: 25 mM Tris, pH 8.0
- Recombinant Human Methionine Aminopeptidase 1D/MAP1D (rhMAP1D) (Catalog # 4714-ZN)
- Recombinant Human DPPIV/CD26 (rhDPPIV/CD26) (Catalog # 9168-SE)
- Substrate: H-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 rhMAP1D to 40 µg/mL in Activation Buffer.
- Dilute Substrate to 500 µM with Activation Buffer.
- Combine equal volumes of 40 µg/mL rhMAP1D with 500 µM Substrate. Include a Substrate Blank containing Activation Buffer and 500 µM Substrate.
- Incubate at room temperature for 1 hour in the dark.
- Stop the reaction by heating for 5 minutes at 95 °C. Put on ice for 3 minutes. Centrifuge briefly in a microcentrifuge.
- Dilute rhDPPIV/CD26 to 2 ng/µL in Assay Buffer.
- Load 50 µL of 2 ng/µL rhDPPIV/CD26 into a plate, and add 50 µL of the rhMAP1D/Substrate mixture (include Substrate Blank).
- Incubate plate at room temperature for 15 minutes in the dark.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
- Calculate specific activity:
|
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 Coumarin (AMC) (Sigma, Catalog # A-9891).
Per Well:- rhMAP1D: 1 µg
- rhDPPIV/CD26: 0.1 µg
- Substrate: 125 µM
Reconstitution Calculator
Background: Methionine Aminopeptidase 1D/MAP1D
Methionine Aminopeptidase 1D (MAP1D) is a member of the Methionine Aminopeptidase family (MetAPs) (1). MetAPs catalyze the removal of the initiator methionine residue from nascent peptides (2) and are essential for cell growth (3). MAP1D is localized in mitochondria (4) and is over-expressed in colon cancer cell lines and colon tumors (1). The N-terminal leader peptide (residue 1-43) of MAP1D has been shown to be responsible for targeting to mitochondria (4).
- Leszczyniecka, M. et al. (2006) Oncogene 25:3471.
- 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.
- Serero, A. et al. (2003) J. Biol. Chem. 278:52953.
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