Recombinant Human alpha-Galactosidase A/GLA Protein, CF

6146-GH Datasheet / COA / SDS

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6146-GH-020

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Summary Product Datasheets Carrier Free Reconstitution Calculator Background Related Research Areas

Recombinant Human alpha-Galactosidase A/GLA Protein, CF Summary

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 hydrolyze 4-methylumbelliferyl-alpha -D-galactopyranoside. The specific activity is >1,100 pmol/min/μg, as measured under the described conditions.

Source

Chinese Hamster Ovary cell line, CHO-derived human alpha-Galactosidase A/GLA protein
Met1-Leu429, with a C-terminal 6-His tag

Accession #

P06280

N-terminal Sequence
Analysis

Leu32

Predicted Molecular Mass

46 kDa

SDS-PAGE

45-55 kDa, reducing conditions

Product Datasheets

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.

6146-GH

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

Assay Buffer: 50 mM Sodium Citrate, 50 mM NaCl, pH 4.0
Recombinant Human alpha ‑Galactosidase A/GLA (rhGLA) (Catalog # 6146-GH)
Substrate: 4-methylumbelliferyl-alpha -D-galactopyranoside (Sigma, Catalog # M7633), 6.7 mM Stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhGLA to 4.0 ng/µL in Assay Buffer.
Dilute Substrate to 800 µM in Assay Buffer.
Load into plate 50 µL of 4.0 ng/µL rhGLA, and start the reaction by adding 50 µL of 800 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate.
Read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
Calculate the 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 4-Methylumbelliferone (Sigma, Catalog # M1381).

Per Well:

rhGLA: 0.200 µg
Substrate: 400 µM

Reconstitution Calculator

Background: alpha-Galactosidase A/GLA

Human alpha -Galactosidase A is a homodimeric glycoprotein that can release terminal alpha -galactosyl moieties from glycolipids and glycoproteins and catalyze the hydrolysis of melibiose into galactose and glucose (1). It is a lysosomal enzyme and is responsible for degradation of glycolipid globotriaosylceramide (Gb3) (Gal alpha 1‑4Gal beta 1‑4Glc beta ‑ceramide). Mutations in this gene cause Fabry disease, an X-linked hereditary lysosomal storagedisease with the accumulation of Gb3 in the walls of small blood vessels,nerves, dorsal root ganglia, renal glomerular and tubular epithelialcells, and cardiomyocytes (2, 3). Inability to prevent the glycosphingolipid deposition can cause hypertension, strokes, heart attack and progressive renal failure (4). Current treatment for Fabry disease is enzyme replacement therapy using intravenously delivered recombinant alpha -Galactosidase A (5, 6).

References

Ioannou, Y.A. et al. (1998) Biochem. J. 332:789.
Koide, T. et al. (1990) FEBS Lett. 259:353.
Ioannou Y.A, et al. (1992) J. Cell Biol. 119:1137.
Germain, D.P. (2002) Expert. Opin. Investig. Drugs. 11:1467.
Barngrover, D. (2003) J. Biotechnol. 95:280.
Mignani, R. and Cagnoli, L. (2004) J. Nephrol. 17:354.

Entrez Gene IDs

2717 (Human)

Alternate Names

agalsidase alfa; Agalsidase alpha; Agalsidase; Alpha-D-galactosidase A; alpha-D-galactoside galactohydrolase 1; Alpha-D-galactoside galactohydrolase; alpha-gal A; alpha-galactosidase A; EC 3.2.1; EC 3.2.1.22; GALA; galactosidase, alpha; GLA; Melibiase