Recombinant F. meningosepticum Endo F1 Protein, CF

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

Recombinant F. meningosepticum Endo F1 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 F. meningosepticum
  Endo F1 in the delycoslyation of other substrates may require alternative conditions for optimal performance.
• Source: E. coli-derived f. meningosepticum Endo-beta-N-acetylglucosaminidase F1/Endo F1 protein
  Arg31-Trp339, with an N-terminal Met and 6-His tag
  Accession # P36911
• Accession #: P36911
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 35 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: 35 kDa, under 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 and NaCl.
• 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 F. meningosepticum Endo-beta -N-acetylglucosaminidase F1/Endo F1 (rFmEndo F1) (Catalog # 5220-GH)
  • Substrate: RNase B (Sigma, Catalog # R1153), 1 mg/mL stock in 10 mM Sodium Phosphate, pH 5.5
  • 15% SDS-PAGE gel
  • Reducing SDS-PAGE gel loading buffer
  • Silver Staining reagents
  • BioRad GS-800 densitometer (or equivalent)
  1. Dilute rFmEndo F1 to 2.5, 0.625, 0.156. 0.039, 0.0098, 0.0024, and 0.0006 μg/mL in Assay Buffer.
  2. Dilute Substrate to 100 μg/mL in Assay Buffer.
  3. Combine 10 μL rFmEndo F1 at each dilution with 10 μL of 100 μg/mL Substrate. Include a control containing 10 μL Assay Buffer and 10 μL of 100 μg/mL Substrate.
  4. Incubate the reaction and control at 37 °C for 30 minutes.
  5. Add 20 μL of reducing gel buffer to each reaction. Boil sample at 100 °C for 3 to 5 minutes before loading to gel.
  6. Load all of the sample (40 μL) per lane on a 15% gel. As a staining development control load 25 ng BSA.
  7. Perform electrophoresis.
  8. Stain gel with silver stain, stop stain development when 25 ng BSA becomes visible.
  9. Analyze % deglycosylation by densitometry.
  10. Determine the DC₅₀ for rFmEndo F1 by plotting % Substrate deglycosylated vs. amount with 4-PL fitting.
  Per Lane:
  • rFmEndo F1: 25, 6.25, 1.56, 0.39, 0.098, 0.024, and 0.006 ng
  • Substrate: 1 µg

Notes

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

Alternate Names for Recombinant F. meningosepticum Endo F1 Protein, CF

• CDC II-a
• EndobetaNacetylglucosaminidase F1
• Endo-beta-N-acetylglucosaminidase F1

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, Endo-beta -N-acetylglucosaminidases from the same bacterial species, including Endo F1, can be used under native conditions to specifically release particular types of N-glycans (1, 2). Because these glycosidases hydrolyze the chitobiose core ofN-glycans, the released glycan products will contain one GlcNAc residue at their reducing ends with the other GlcNAc residue remaining attached to an asparagine residue on the glycoprotein. Endo F1 specifically releases oligomannose and hybrid but not complex type N-glycans from glycoproteins (3, 4). This enzyme is also suitable to deglycosylate substrates under denaturing conditions and remains active on sulfated and core fucosylated N-glycans at reduced rates (4).

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

Bioinformatics

• Uniprot:
  • P36911()