• Purity

  >95%, 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.  

• Activity

  Measured by its ability to deglycosylate TSLP under native conditions. The DC    ₅₀ is<12 ng. The DC    ₅₀ is defined as the amount of enzyme required to remove 50% of glycan on 1 μg of recombinant mouse TSLP in 30 minutes at 37 °C. See Activity Assay Protocol on www.RnDSystems.com. Use of Recombinant     F. meningosepticum Endo? beta ?N?acetylglucosaminidase F3/Endo F3 in the delycoslyation of other substrates may require alternative conditions for optimal performance.

• Source

  E. coli-derived Ala44-Asn329, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  32 kDa

• SDS-PAGE

  30 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM NaOAc, pH 4.5

• Recombinant F. meningosepticum Endo F3 (rFmEndo F3) (Catalog # 5548-GH)

• Substrate: Recombinant Mouse TSLP (Catalog # )

• 15% SDS-PAGE gel

• Reducing SDS-PAGE gel loading buffer (≥3X concentration)

• Silver Staining reagents

• BioRad GS-800 densitometer (or equivalent)

1. Dilute rFmEndo F3 to 5, 2.5, 1.25, 0.625. 0.313, 0.156, 0.078 μg/mL in Assay Buffer.

2. Dilute Substrate to 100 μg/mL in Assay Buffer.

3. Combine 10 μL rFmEndo F3 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 10 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 (30 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 rFmEndoF3 by plotting % Substrate deglycosylated vs. amount with 4-PL fitting.

Per Lane:

• rFmEndo F3: 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 ng

• Substrate: 1 μg

Background: Endo-beta-N-acetylglucosaminidase F3/Endo F3

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 can be used under native conditions to specifically release particular types of N-glycans (1, 2). Because these glycosidases hydrolyze the chitobiose core of N-glycans, the released glycan products will contain one GlcNAc residue at their reducing ends with the other GlcNAc residue remaining attached to the asparagine residue on the glycoproteins. Endo F3 hydrolyzes bi- and triantennary N-glycans (3). In general, this enzyme is more efficient on core fucosylated N-glycans increasing activity up to 400-fold with biantennary structures (4). It also cleaves fucosylated trimannosyl core structures.

• References:

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. (1993) J. Biol. Chem. 268: 9702.

4. Tarentino, A.L. and Plummer, T.H. Jr. (1994) Glycobiology 4:771.

• Alternate Names:

  Endo F3; EndobetaNacetylglucosaminidase F3; Endo-beta-N-acetylglucosaminidase F3