Home / Endo-beta-N-acetylglucosaminidase F3/Endo F3 / Recombinant F. meningosepticum Endo F3 Protein, CF

Recombinant F. meningosepticum Endo F3 Protein, CF

Catalog #: 5548-GH Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
5548-GH-010
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Recombinant F. meningosepticum Endo F3 Protein, CF Summary

Product Specifications

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 DC50 is <12 ng. The DC50 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.. 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 f. meningosepticum Endo-beta-N-acetylglucosaminidase F3/Endo F3 protein
Ala44-Asn329, with an N-terminal Met and 6-His tag
Accession #
P36913
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
32 kDa
SDS-PAGE
30 kDa, reducing conditions

Product Datasheets

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5548-GH

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

5548-GH

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.

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 # 555-TSB)
  • 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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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

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