Recombinant Human A4GNT Protein, CF

Catalog #: 8960-GT Datasheet

Catalog #	Availability	Size / Price	Qty
8960-GT-020

R&D Systems Recombinant Proteins and Enzymes

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All Alpha-1,4-N-Acetylglucosaminyltransferase 4/A4GNT Proteins and Enzymes

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Recombinant Human A4GNT Protein, CF Summary

Product Specifications

Purity

>95%, 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

Measured by its ability to transfer GlcNAc from UDP-GlcNAc to galactose. The specific activity is >400 pmol/min/μg, as measured under the described conditions.

Source

Chinese Hamster Ovary cell line, CHO-derived human Alpha-1,4-N-Acetylglucosaminyltransferase 4/A4GNT protein
Leu25-Lys340, with C-terminal 6-His tag

Accession #

Q9UNA3

N-terminal Sequence
Analysis

Leu25

Predicted Molecular Mass

38 kDa

SDS-PAGE

39-49 kDa, reducing conditions

Product Datasheets

8960-GT

Product Datasheet

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.

8960-GT

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Glycosyltransferase Activity Kit (Catalog # EA001)
10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl₂, pH 7.5
MnCl₂ (supplied in kit): 100 mM
Recombinant Human alpha -1,4-N-Acetylglucosaminyltransferase 4/A4GNT (rhA4GNT) (Catalog # 8960-GT)
UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
D-(+)-Galactose (Sigma, Catalog # G0625), 750 mM stock in deionized water
96-well Clear Plate (Catalog # DY990)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining 10X stocks and diluting 10-fold with deionized water.
Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
Prepare reaction mixture containing 5 mM UDP-GlcNAc, 200 mM D-(+)-Galactose, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
Dilute rhA4GNT to 4 ng/µL in 1X Assay Buffer.
Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
Load 25 µL of 4 ng/µL rhA4GNT into empty wells of the same plate as the curve. Include a Control containing 25 μL of 1X Assay Buffer.
Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
Seal plate and incubate at 37°C for 20 minutes.
Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
Add 100 µL of deionized water to all wells. Mix briefly.
Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
Read plate at 620 nm (absorbance) in endpoint mode.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Phosphate released* (nmol) x (1000 pmol/nmol)
	Incubation time (min) x amount of enzyme (µg)

*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

rhA4GNT: 0.1 µg
Coupling Phosphatase 1: 0.1 µg
UDP-GlcNac: 2.5 mM
D-(+)-Galactose: 100 mM

Background: Alpha-1,4-N-Acetylglucosaminyltransferase 4/A4GNT

Alpha-1,4-N-acetylglucosaminyltransferase A4GNT is a type II Golgi resident membrane glycosyltransferase, but shows no significant sequence similarity with any other glycosyltransferases. It catalyzes the transfer of N-acetylglucosamine (GlcNAc) to core 2 branched O-glycans and forms a unique glycan,
GlcNAc alpha 1-->4Gal beta -->R on mucin (1). Alpha-1,4-GlcNAc-capped mucin-type O-glycan inhibits cholesterol alpha -glucosyltransferase from Helicobacter pylori (Hp) and suppresses Hp growth (2). Hp infection is widespread and thought to be the causes of the development of gastric lesions including gastritis, intestinal metaplasia, and gastric carcinoma (3). The enzymatic activity of the recombinant protein was determined using a phosphatase-coupled assay (4).

References