Recombinant E. coli Methionine Aminopeptidase/METAP, CF

Catalog #: 1628-ZN Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
1628-ZN-020

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Recombinant E. coli Methionine Aminopeptidase/METAP, CF Summary

Product Specifications

Purity

>85%, 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

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 >0.5 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com.

Source

E. coli-derived e. coli Methionine Aminopeptidase protein
Ala2-Glu264, with a C-terminal 10-His tag

Accession #

NP_414710

N-terminal Sequence
Analysis

Ala2

Predicted Molecular Mass

31 kDa

SDS-PAGE

36 kDa, reducing conditions

Product Datasheets

1628-ZN

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.

1628-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 E. coli Methionine Aminopeptidase (reMAP) (Catalog # 1628-ZN)
Recombinant Human DPPIV/CD26 (rhDPPIV) (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 reMAP to 40 µg/mL in Activation Buffer.
Dilute Substrate to 200 µM in Activation Buffer.
Mix 100 µL of 40 µg/mL reMAP with 100 µL 200 µM Substrate. Include a Substrate Blank containing 100 µL of Activation Buffer and 100 µL 200 µM Substrate.
Incubate at 37 °C for 15 minutes.
Stop the reaction by heating for 5 minutes at 95 to 100 °C.
Place reactions on ice for 3 minutes, then spin briefly.
Dilute rhDPPIV/CD26 to 2 µg/mL in Assay Buffer.
Load 50 µL of reaction mixture into a plate and add 50 µL of 2 µg/mL rhDPPIV/CD26.
Incubate plate with shaking for 10 minutes at room temperature.
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 # A9891).

Per Well:

reMAP: 1 µg
rhDPPIV/CD26: 0.100 µg
Substrate: 50 µM

Reconstitution Calculator

Background: Methionine Aminopeptidase

Methionine Aminopeptidase (METAP) carries out the important reaction of removing the initiator Met residue from nascent proteins. There are two types of METAP (1, 2). Type Ia and type IIa are found in eubacteria and archaea, respectively. Type Ib and type IIb are present in eukaryotes and contain additional N-terminal domains. As a metalloenzyme, METAP activity can be inhibited by EDTA. However, the exact identity of the metal ion remains to be uncovered since several metal ions including cobalt, zinc and iron have been suggested (3).

References