Recombinant Human g-Glutamylcyclotransferase/CRF21, CF

Catalog #: 5086-CT Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
5086-CT-010

R&D Systems Recombinant Proteins and Enzymes

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All gamma-Glutamylcyclotransferase/CRF21 Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human g-Glutamylcyclotransferase/CRF21, 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 release L-alanine from gamma -glutamyl-L-alanine, with the detection of alanine by alanine dehydrogenase. The specific activity is >40,000 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived human gamma-Glutamylcyclotransferase/CRF21 protein
Met1-Leu188

Accession #

O75223

N-terminal Sequence
Analysis

Ala2

Predicted Molecular Mass

21 kDa

SDS-PAGE

20-22 kDa, reducing conditions

Product Datasheets

5086-CT

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.

5086-CT

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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, pH 9.0
Recombinant Human gamma ‑Glutamylcyclotransferase/CRF21 (rhCRF21) (Catalog # 5086-CT)
gamma -L-Glutamyl-L-alanine (Sigma, Catalog # 483834), 100 mM stock in deionized water
beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
L-Alanine Dehydrogenase (Sigma, Catalog # A7653)
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Dilute rhCRF21 to 0.05 ng/μL in Assay Buffer.
Prepare a Substrate Mixture containing 10 mM gamma -L-Glutamyl-L-alanine, 2 mM beta -NAD, and 0.008 units/μL L‑Alanine Dehydrogenase in Assay Buffer.
Load into a plate 50 μL of 0.05 ng/μL rhCRF21, and start the reaction by adding 50 μL of Substrate Mixture. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
Read plate at a wavelength of 340 nm (bottom read) in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (OD/min) x well volume (L) x 10¹² pmol/mol
	ext. coeff (M^-1cm^-1) x path corr.* (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Using the extinction coefficient 6220 M^-1cm^-1
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD.

hCRF-21: 0.0025 μg
gamma -L-Glutamyl-L-alanine: 5 mM
beta -NAD: 1 mM
L-Alanine Dehydrogenase: 0.4 units

Reconstitution Calculator

Background: gamma-Glutamylcyclotransferase/CRF21

Gamma-glutamylcyclotransferase (GGCT) is an essential enzyme in the gamma -glutamyl cycle that catalyzes the formation of 5-oxoproline from gamma-glutamyl dipeptides. The enzyme plays an important role in glutathione homeostasis. GGCT is also known as cytochrome c-releasing factor 21(CRF-21). It induces the release of cytochrome c from mitochondria, leading to apoptosis (1). It is a 188 amino acid protein that consists of six beta -strands, five alpha -helices and four short 3₁₀helices (2). Up-regulation of the expression of GGCT has been found in various tumor tissues including lung, esophagus, stomach, bile duct, uterine cervix, colon and breast. (3, 4). Therefore, GGCT is a potential biomarker for numerous types of cancers.

References