Recombinant Rat Insulysin/IDE Protein, CF

Catalog #: 6958-ZN Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
6958-ZN-010

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Rat Insulysin/IDE Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ES005). The specific activity is >1,600 pmol/min/μg, as measured under the described conditions.

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived rat Insulysin/IDE protein
Met42-Leu1019, with an N-terminal Met and 5-His tag

Accession #

P35559

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

114 kDa

SDS-PAGE

95-115 kDa, reducing conditions

Product Datasheets

6958-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.

6958-ZN

Formulation	Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, and Brij-35.
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

Assay Buffer: 50 mM Tris, 1 M NaCl pH 7.5
Recombinant Rat Insulysin/IDE (rrInsulysin) (Catalog # 6958-ZN)
Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005), 2 mM in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rrInsulysin to 0.2 µg/mL in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of the 0.2 µg/mL rrInsulysin into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

rrInsulysin: 0.01 µg
Substrate: 10 µM

Reconstitution Calculator

Background: Insulysin/IDE

Insulysin, or insulin-degrading enzyme (IDE), is a zinc metallopeptidase of the inverzincin family. IDE is primarily located in the cytosol, but has been detected as a secreted enzyme and associated with the plasma membrane as well (1). The enzyme is expressed in many tissues, with the highest levels in liver, kidney, brain, and testis (2). IDE hydrolyzes a variety of regulatory peptides, including insulin, glucagon, atrial natriuretic factor, and transforming growth factor-a in vitro (1). In addition, IDE has been shown to degrade the amyloid b (Ab) peptide, which polymerizes into the plaques associated with Alzheimer’s disease (3). Deficiencies in IDE activity may contribute to the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer’s disease. The IDE region of human chromosome 10q has been genetically linked to DM2 (4). When the IDE gene was specifically disrupted in mice, IDE -/- animals developed hyperinsulinemia and glucose intolerance, characteristics of DM2 (5). The IDE -/- mice were also shown to have a significant decrease in Ab degradation in the brain, resulting in increased cerebral accumulation of Ab peptide (6). This in vivo evidence is consistent with the hypotheses that IDE is important for the degradation of insulin in cells and for the clearance of Ab peptide in the brain. Rat Insulysin displays 95.5% and 98.5 % sequence homology with human and mouse insulysin, respectively.

References