Recombinant F. meningosepticum PNGase F Protein, CF

Name: Recombinant F. meningosepticum PNGase F Protein, CF
Brand: R&D Systems
SKU: 7695-GH

Catalog #: 7695-GH Datasheet / COA / SDS

Newer Version Available: 9109-GH

NEW

Discontinued Product

7695-GH has been discontinued and is replaced by 9109-GH.

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Recombinant F. meningosepticum PNGase F Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

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 denatured conditions. >50% ribonuclease B (10 μg) is deglycosylated by 10 ng of rFmPNGase F within 30 minutes, as measured under the described conditions.

Source

E. coli-derived f. meningosepticum PNGase F protein
Ala41-Asn354 with N-terminal Met and 6-His tag

Accession #

AAA85323

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

36 kDa

SDS-PAGE

33-35 kDa, reducing conditions

Product Datasheets

7695-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.

7695-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: 0.1 M Tris, pH 7.5
Denaturing Buffer (10X): 5% SDS, 0.8 M beta -Mercaptoethanol
Recombinant F. meningosepticum PNGase F (rFmPNGaseF) (Catalog # 7695-GH)
Ribonuclease B, from bovine pancreas (RNase B) (Sigma, Catalog # R7884), 2.5 mg/mL stock in 25 mM Tris, pH 7.5
10% (v/v) Triton® X-100 in deionized water
Reducing SDS-PAGE Sample Buffer
SDS-PAGE or Western Blot

Dilute Denaturing Buffer to 5X in deionized water.
Create a Substrate Mixture containing 0.8 mg/mL RNAse B and 1X denaturing buffer in deionized water.
Heat Substrate Mixture at 100 °C for 10 minutes. Cool to room temperature and microcentrifuge briefly.
Add Triton X-100 to a final concentration of 1.67%.
Dilute rFmPNGaseF to 0.67 ng/µL in Assay buffer.
Combine 15 µL of Substrate Mixture and 15 µL of 0.67 ng/µL rFmPNGaseF. Include a control containing 15 µL of Substrate Mixture and 15 µL of Assay buffer.
Incubate at 37 °C for 30 minutes.
Combine equal volumes of incubated reaction mixture and reducing SDS-PAGE sample buffer and boil samples at 100 °C for 3‑5 minutes.
Load 15 µL (2.5 µg RNase B) per lane on a 4-20% SDS-PAGE gel.
Stain gel and analyze for percent deglycosylation using densitometry.

Per Reaction:

rFmPNGaseF: 10 ng
RNase B: 10 µg

Reconstitution Calculator

Background: PNGase F

PNGase F, peptide N-glycosidase F from Flavobacterium meningosepticum, catalyzes the hydrolysis of asparagine-linked high mannose, as well as hybrid and complex oligosaccharides from glycoproteins (1). Unlike glycosidases that hydrolyze glycosidic bonds, PNGase F is an amidase that cleaves the beta ‑aspartylglucosamine bond between the innermost GlcNAc of N-glycans and asparagine residues of glycoproteins (2). The enzyme is highly active on various N‑glycans except those with the innermost GlcNAc modified with alpha 1-3-linked core fucose, which is commonly found on plant glycoproteins (3). Cleavage with PNGase F will convert the asparagine residue to an aspartic residue, allowing identification of the glycosylation site by mass spectrometry (4). This purified enzyme is compatible with glycan analysis using mass spectrometry.

References