Recombinant F. meningosepticum Endo F1 Protein, CF

Catalog #: 5220-GH Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
5220-GH-050

R&D Systems Recombinant Proteins and Enzymes

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All Endo-beta-N-acetylglucosaminidase F1/Endo F1 Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant F. meningosepticum Endo F1 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 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
Analysis

Met

Predicted Molecular Mass

35 kDa

SDS-PAGE

35 kDa, under reducing conditions

Product Datasheets

5220-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.

5220-GH

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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 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)

Dilute rFmEndo F1 to 2.5, 0.625, 0.156. 0.039, 0.0098, 0.0024, and 0.0006 μg/mL in Assay Buffer.
Dilute Substrate to 100 μg/mL in Assay Buffer.
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.
Incubate the reaction and control at 37 °C for 30 minutes.
Add 20 μL of reducing gel buffer to each reaction. Boil sample at 100 °C for 3 to 5 minutes before loading to gel.
Load all of the sample (40 μL) per lane on a 15% gel. As a staining development control load 25 ng BSA.
Perform electrophoresis.
Stain gel with silver stain, stop stain development when 25 ng BSA becomes visible.
Analyze % deglycosylation by densitometry.
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

Reconstitution Calculator

Background: Endo-beta-N-acetylglucosaminidase F1/Endo F1

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).

References