Recombinant Human a-N-acetylgalactosaminidase Protein, CF
Recombinant Human a-N-acetylgalactosaminidase Protein, CF Summary
Product Specifications
Leu18-Gln411, with a C-terminal 6‑His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
6717-GH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij and Glycerol. |
| 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.
|
Assay Procedure
- Assay Buffer: 0.1 M Sodium Citrate, 0.2 M NaCl, pH 4.0
- Recombinant Human alpha ‑N‑acetylgalactosaminidase/NAGA (rhNAGA) (Catalog # 6717-GH)
- Substrate: 4-Nitrophenyl-N-acetyl-alpha -D-galactosaminide (Sigma, Catalog # N4264), 15 mM stock in DMSO
- 0.2 M Sodium Hydroxide
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhNAGA to 2 ng/μL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load 50 µL of 2 ng/µL rhNAGA and 50 µL of 2 mM Substrate into a clear 96-well plate. Also create a Substrate Blank by loading 50 µL of Assay Buffer and 50 µL of 2 mM Substrate.
- Incubate plate at room temperature for 10 minutes.
- Add 100 μL of 0.2 M NaOH to each well used in order to stop the reaction and develop the color.
- Read absorbance in endpoint mode at 402 nm.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Abs* (OD) x volume† (L) x 1012 pmol/mol |
| Inc. time (min) x epsilon ** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 17700 M-1cm-1
***Using the path corr. 0.6 cm
†Based upon a 0.0002 L Volume
- rhNAGA: 0.1 μg
- Substrate: 1 mM
Reconstitution Calculator
Background: alpha-N-acetylgalactosaminidase/NAGA
NAGA is a lysosomal alpha -N-acetylgalactosaminidase that cleaves non-reducing alpha -N-acetylgalactosaminyl moieties from glycoconjugates (1). Mature NAGA has 394 amino acids and is trafficked to the lysosome via the mannose-6-phosphate receptor‑mediated pathway (2). The enzyme is a retaining exoglycosidase, where both the substrate and product of the enzymatic reaction have the same anomeric configuration (3). Deficiency in NAGA results in increased urinary excretion and tissue accumulation of glycopeptides and oligosaccharides containing terminal alpha ‑N‑acetylgalactosaminyl moieties (4), manifesting as Schindler’s disease, an autosomal recessive disease with neuroaxonal dystrophy and other neurological symptoms (5). The enzyme can be used to remove alpha ‑N‑acetylgalactosaminyl residues present on red blood cells thus converting blood type A to blood type O (6, 7, 8).
- Wang, A.M. et al. (1990) J. Biol. Chem. 265:21859.
- Sweeley, C.C. et al. (1983) Arch. Biochem. Biophys. 223:158.
- Garman, S.C. et al. (2002) Structure. 10:425.
- Eng, C.M. et al. (2001) N. Engl. J. Med. 345:9.
- Wang, A.M. et al. (1990) J. Clin. Invest. 86:1752.
- Liu, Q.P. et al. (2007) Nature Biotechnol. 25:454.
- Calcutt, M. J. et al. (2002) FEMS Microbiol. Lett. 214:77.
- Zhu, A. et al. (1996) Arch. Biochem. Biophys. 327:324.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citation for Recombinant Human a-N-acetylgalactosaminidase Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates
Authors: S Cecioni, RA Ashmus, PA Gilormini, S Zhu, X Chen, X Shan, C Gros, MC Deen, Y Wang, R Britton, DJ Vocadlo
Nature Chemical Biology, 2022-02-24;18(3):332-341.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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As a marker PageRuler Prestained Protein Ladder, 10 to 180 kDa (Thermo Scientific, #26616) was used NAGAL was detected using anti-NAGA antibody (Abcam, ab102668, dilution 1/1.000) as primary and anti-rabbit IgG HRP-conjugated [Merck, #12-348, dilution: 1/20.000] as secondary antibody.
Figure legend: Lane 1: 10 ng agalsidase-alfa, no signal; Lane 2: 10 ng recombinant human a-N-acetylgalactosaminidase Protein (RnD Systems,#6717-GH-020), distinct band between 40 -60 kDa as predicted.
