Recombinant S. plicatus Endo H Protein, CF

• Reactivity: Sp
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant S. plicatus Endo H Protein, CF Summary

• Details of Functionality: 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.
• 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
• Accession #: P04067
• N-terminal Sequence: MHHHHHHVKQ
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <0.01 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 30 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 29 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant S. plicatus Endo H Protein, CF

• EndobetaNacetylglucosaminidase H
• Endo-beta-N-acetylglucosaminidase H

Background

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).

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.

Bioinformatics

• Uniprot:
  • P04067()