Recombinant Human a-N-acetylgalactosaminidase Protein, CF

R&D Systems | Catalog # 6717-GH

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human a-N-acetylgalactosaminidase Protein (6717-GH)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

P17050

Applications

Bioactivity
View all alpha-N-acetylgalactosaminidase/NAGA Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human alpha-N-acetylgalactosaminidase/NAGA protein
Leu18-Gln411, with a C-terminal 6‑His tag

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.

N-terminal Sequence Analysis

Leu18

Predicted Molecular Mass

46 kDa

SDS-PAGE

50-60 kDa, reducing conditions

Activity

Measured by its ability to cleave alpha -N-acetylgalactosaminyl from 4-Nitrophenyl N-acetyl-alpha -D-galactosaminide.
The specific activity is >1800 pmol/min/μg, as measured under the described condition.

Reviewed Applications

Read 1 review rated 5 using 6717-GH in the following applications:

Formulation, Preparation, and Storage

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

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).

References

  1. Wang, A.M. et al. (1990) J. Biol. Chem. 265:21859.
  2. Sweeley, C.C. et al. (1983) Arch. Biochem. Biophys. 223:158.
  3. Garman, S.C. et al. (2002) Structure. 10:425.
  4. Eng, C.M. et al. (2001) N. Engl. J. Med. 345:9.
  5. Wang, A.M. et al. (1990) J. Clin. Invest. 86:1752.
  6. Liu, Q.P. et al. (2007) Nature Biotechnol. 25:454.
  7. Calcutt, M. J. et al. (2002) FEMS Microbiol. Lett. 214:77.
  8. Zhu, A. et al. (1996) Arch. Biochem. Biophys. 327:324.

Alternate Names

alphaNacetylgalactosaminidase, NAGA

Entrez Gene IDs

4668 (Human); 17939 (Mouse); 315165 (Rat)

Gene Symbol

NAGA

UniProt

P17050

Additional alpha-N-acetylgalactosaminidase/NAGA Products

Product Documents for Recombinant Human a-N-acetylgalactosaminidase Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human a-N-acetylgalactosaminidase Protein, CF

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

For research use only

Citations for Recombinant Human a-N-acetylgalactosaminidase Protein, CF

Customer Reviews for Recombinant Human a-N-acetylgalactosaminidase Protein, CF (1)

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  • Recombinant Human a-N-acetylgalactosaminidase Protein, CF
    Name: Anonymous
    Application: Western Blot
    Verified Customer | Posted 09/09/2019
    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.
    Recombinant Human a-N-acetylgalactosaminidase Protein, CF 6717-GH

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Protocols

View specific protocols for Recombinant Human a-N-acetylgalactosaminidase Protein, CF (6717-GH):

Materials
  • 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
  1. Dilute rhNAGA to 2 ng/μL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. 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.
  4. Incubate plate at room temperature for 10 minutes.
  5. Add 100 μL of 0.2 M NaOH to each well used in order to stop the reaction and develop the color.
  6. Read absorbance in endpoint mode at 402 nm.
  7. 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

Per Reaction:
  • rhNAGA: 0.1 μg
  • Substrate: 1 mM

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