Recombinant S. plicatus Endo H Protein, CF

R&D Systems | Catalog # 5224-GHB

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Key Product Details

  • R&D Systems E. coli-derived Recombinant S. plicatus Endo H Protein (5224-GHB)
  • 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

P04067

Applications

Enzyme Activity
View all Endo-beta-N-acetylglucosaminidase H/Endo H Proteins and Enzymes »

Product Specifications

Source

E. coli-derived s. plicatus Endo-beta-N-acetylglucosaminidase H/Endo H protein
Val47-Pro313 with an N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<0.01 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

MHHHHHHVKQ

Predicted Molecular Mass

30 kDa

SDS-PAGE

29 kDa, reducing conditions

Activity

Measured by its ability to deglycosylate ribonuclease B under native conditions.
The DC50 is <5 ng. The DC50 is defined as the amount of enzyme required to remove 50% of glycan on 1 μg of RNase B in 30 minutes at
37 °C.. Use of Recombinant S. plicatus Endo-beta -N-acetylglucosaminidase H/Endo H in the deglycoslyation of other substrates may require alternative conditions for optimal performance.

Formulation, Preparation, and Storage

5224-GHB
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA.
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: Endo-beta-N-acetylglucosaminidase H/Endo H

N-glycans are commonly found on various glycoproteins. While peptide N-glycosidase from Flavobacterium meningosepticum (PNGase F) is widely used to release virtually all types of N-glycans under denaturing conditions, some bacterial endo-beta -N-acetylglucosaminidases, including Endo H, can be used under native conditions to specifically release particular types of N-glycans (1, 2). Because these glycosidases hydrolyze the chitobiose core of an N-glycan on a glycoprotein, the released glycan product will contain one GlcNAc residue at its reducing end with the other GlcNAc residue remaining attached to the glycoprotein. Endo H specifically releases oligomannose and hybrid but not complex type N-glycans from glycoproteins (3-5). It also remains active on core fucosylated N-glycans but not sulfated glycans (5).

References

  1. Maley, F. et al. (1989) Anal. Biochem. 180:195.
  2. Tarentino, A.L. et al. (1985) Biochemistry 24:4665.
  3. Hsieh, P. et al. (1983) J. Biol. Chem. 258:2555.
  4. Tarentino, A.L. et al. (1992) J. Biol. Chem. 267:3868.
  5. Trimble, R.B. and Tarentino, A.L. (1991) J. Biol. Chem. 266:1646.

Alternate Names

EndobetaNacetylglucosaminidase H

UniProt

P04067

Additional Endo-beta-N-acetylglucosaminidase H/Endo H Products

Product Documents for Recombinant S. plicatus Endo H 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 S. plicatus Endo H Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant S. plicatus Endo H Protein, CF (5224-GHB):

Materials
  • Assay Buffer: 50 mM Sodium Phosphate, pH 5.5
  • Recombinant S. plicatus Endo-beta -N-acetylglucosaminidase H/Endo H (rS. plicatus Endo H) (Catalog # 5224-GHB)
  • RNase B (Sigma, Catalog # R1153), 1 mg/mL stock in 10 mM Sodium Phosphate, pH 5.5
  • 4-20% SDS-PAGE gel
  • Reducing SDS-PAGE gel loading buffer
  • Silver Staining reagents
  1. Dilute Endo H to 2.5, 1.25, 0.625. 0.313, 0.156, 0.0781, and 0.0391 μg/mL in Assay Buffer.
  2. Dilute RNase B to 100 μg/mL in Assay Buffer.
  3. Combine 25 μL of Endo H at each dilution with 25 μL of 100 μg/mL RNase B. Include a control containing 25 μL Assay Buffer and 25 μL of 100 μg/mL RNase B.
  4. Incubate the reaction and control at 37 °C for 30 minutes.
  5. Combine 30 μL of  each reaction with 30 μL of reducing SDS-PAGE gel loading buffer. Heat samples at 100 °C for 3 to 5 minutes before loading to gel.
  6. Load 40 μL of sample per lane (1 μg of RNase B per lane) on a 15% gel.
  7. Stain gel with silver stain.
  8. Determine the 50% deglycosylation concentration (DC50) for Endo H by plotting % of RNase B deglycosylated vs. Endo H concentration with 4-PL fitting.
Per Lane:
  • rS. plicatus Endo H: 25, 12.5, 6.25, 3.13, 1.56, 0.781, and 0.391 ng
  • RNase B: 1 µg

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