N-Glycanase-PLUS® (PNGase F), 1 U [GKE-5010D]

Price:

Description

PNGase F, peptide-N-glycosidase F, peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase. Releases intact N-glycans by cleaving between the innermost GlcNAc and Asn.

Source:
Recombinant gene from Elizabethkingia meningosepticum, expressed in E. coli. The source organism was previously known as Chryseobacterium [Flavobacterium] meningosepticum [1]. 

Specificity: 
Cleaves all N-linked complex, hybrid or high mannose oligosaccharides, unless α(1-3) core fucosylated, as in plant glycans. Asparagine must be peptide-bonded at both termini. Phosphate, sulfate and sialic acid groups attached to the oligosaccharide do not affect cleavage. Endo F free. Highly concentrated, particularly useful for deglycosylation under native conditions. 

 

Figure 1: Cleavage specificity for GKE-5010 N-Glycanase (PNGase F).
R1: N & C substitution other than H
R2: may be H or the rest of an oligosaccharide
R3: may be H or α(1-6)-linked fucose

Applications: 
To obtain efficient deglycosylation of glycoprotein substrates under non-denaturing conditions, it is necessary to use a higher starting concentration of enzyme. N-Glycanase® PLUS and ULTRA (EDTA-Free) are supplied at ≥ 10 U/ml, and are recommended for all applications requiring deglycosylation of glycoproteins in the absence of denaturants. The high activity also allows smaller reaction volumes and shorter reaction times to be explored.

Note: the "D" 1 U pack size does not ship with reaction buffers, as does the GKE-5010B pack size, although reaction buffers are available on request.

Formulation:
20 mM Tris HCl pH 7.5, containing 1 mM EDTA and 50 mM NaCl

pH optimum:
8.6

pH range:
7.5 - 9.5

Unit Definition:
One unit is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 μmole of denatured ribonuclease B per minute at pH 7.5 and 37°C.

Size: 1 U (100 µl)

Concentration: ≥ 10 U/ml

Product Code: GKE-5010D

Not formulated for FACE® Analysis, use GK50052.

1. Kim KK, Kim MK, Lim JH, Park HY, Lee ST. Transfer of Chryseobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen. nov. as Elizabethkingia meningoseptica comb. nov. and Elizabethkingia miricola comb. nov.  2005 May;55(Pt 3):1287-93.

Tech Info

GKE-5010D Technical Data Sheet  

FAQs

Q. You have a number of PNGase F products. What are the differences?

A. We carry a number of different formulations and pack sizes of PNGase F:

Product Pack size Volume Concentration* Recombinant Native EDTA
GKE-5006 A - 100 mU 40 µl ≥ 2.5 U/ml 1mM
B - 200 mU 80 µl
D - 1 U† 400 µl
GKE-5016 A - 100 mU 40 µl ≥ 2.5 U/ml 0
B - 200 mU 80 µl
D - 1 U 400 µl
GKE-5010 (PLUS) B - 400 mU 40 µl ≥ 10 U/ml 1 mM
D - 1 U 100 µl
GKE-5020 (ULTRA) B - 400 mU 40 µl ≥ 10 U/ml 0
D - 1 U 100 µl
GKE-5003 100 mU 50 µl ≥ 2.0 U/ml 5 mM

*One unit of N-Glycanase is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 μmole of denatured ribonuclease B per minute at pH 7.5 and 37°C.

†Buffers and denaturant are not supplied with 1 U "D" pack sizes, but can be made available on request.

EDTA was included historically in native PNGase F formulations as a stabilizer. For the GKE-5020 formulation we revisited the idea, due to requests from customers for a PNGase F formulation without EDTA. We believe that stability is not as much of a concern with the recombinant form of the enzyme.

Q. I am running MS of proteins with and without PNGase F treatment. Is there a +1 Da change when using PNGaseF in addition to the MW loss of the N-linked glycan?

A. Yes, a + 1 Da change on the protein is to be expected when deglycosyalting with PNGase F. The process of PNGase F removing the N-glycans involves the deamination of asparagine to aspartic acid. We have some references on this in our tech data sheet for PNGase F.

Asparagine (N): 132.12 Da

Aspartic acid (D): 133.10 Da

Safety

Product Safety Documentation for GKE-5010D:

Product/Part No. Description
GKE-5010D Glyko® N-Glycanase-PLUS 

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