Recombinant C. perfringens a-N-acetylgalactosaminidase, CF

R&D Systems | Catalog # 5705-GH

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Key Product Details

  • R&D Systems E. coli-derived Recombinant C. perfringens a-N-acetylgalactosaminidase (5705-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

E. coli

Accession Number

AAM55479

Applications

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

Product Specifications

Source

E. coli-derived c. perfringens alpha-N-acetylgalactosaminidase/NAGA protein
Lys2-Lys619 with an N-terminal Met and 6-His tag
Accession # AAM55479

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

75 kDa

SDS-PAGE

65 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 >5,000 pmol/min/ug, as measured under the described conditions.

Formulation, Preparation, and Storage

5705-GH
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.

Background: alpha-N-acetylgalactosaminidase/NAGA

The ABO blood group system is the most important blood type system and alpha -N-acetylgalactoside is key to the ABO blood group antigen (1). alpha -N-acetylgalactosidase from Clostridium perfringens is a useful tool for removing alpha linked N-acetylgalactosamine from blood type A antigen to produce H antigen and blood type O (2, 3). Blood type O is universally compatible in the ABO system and is widely used in blood transfusion (2). The enzyme was highly selective for terminal N‑acetylgalactosamine residues (3). No other exoglycosidase activities, particularly neuraminidase, was detected. Recombinant alpha -N-acetylgalactosidase from Clostridium perfringens can potentially be used in enzymatic conversion of human blood type A red blood cells to universally transfusable type O red blood cells.

References

  1. Watkins, W.M. (1980) Adv. Hum. Genet. 10:1.
  2. Liu, Q.P. et al. (2007) Nature Biotechnol. 25:454.
  3. Calcutt, M. J. et al. (2002) FEMS Microbiol. Lett. 214:77.
  4. Hsieh, H.Y. et al. (2003) Protein Expr. Purif. 32:309.

Alternate Names

alphaNacetylgalactosaminidase, NAGA

Entrez Gene IDs

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

Gene Symbol

NAGA

UniProt

AAM55479

Additional alpha-N-acetylgalactosaminidase/NAGA Products

Product Documents for Recombinant C. perfringens a-N-acetylgalactosaminidase, 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 C. perfringens a-N-acetylgalactosaminidase, CF

For research use only

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Protocols

View specific protocols for Recombinant C. perfringens a-N-acetylgalactosaminidase, CF (5705-GH):

Materials
  • Assay Buffer: 100 mM MES, pH 6.5
  • Recombinant C. perfringens alpha -N-acetylgalactosaminidase (rC. perfringens alpha -N-galactosaminidase) (Catalog # 5705-GH)
  • Substrate: 4-Nitrophenyl N-acetyl-alpha-D-galactosaminide (Sigma-Aldrich, Catalog # N4264)
  • 0.2 M NaOH, prepare in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rC. perfringens alpha -N-galactosaminidase to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Combine 50 µL of alpha -N-galactosaminidase and 50 µL of Substrate in wells of a 96-well plate. Substitute alpha -N-galactosaminidase with Assay Buffer for Substrate Blank.
  4. Incubate at room temperature for 5 minutes.
  5. Stop the reaction by adding 100 µL of 0.2 M NaOH, which also develops the color.
  6. Read at 402 nm in endpoint mode.
  7. Calculate specific activity:

     Specific Activity (pmoles/min/µg) =

 Adjusted Abs* (OD) x well volume (L) x 1012 pmol/M
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 correction 0.600 cm

Per Well:
  • rC. perfringens alpha -N-galactosaminidase: 0.05 µg
  • Substrate: 0.5 mM

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