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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.
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
The enzyme can be used with sodium dodecyl sulfate (SDS) and β-mercaptoethanol (βME) at certain levels.
The GK80110 Enzymatic Deglycosyaltion Kit uses SDS and bME in the denaturing protocol.
The 5x Denaturation Solution used in the GK80110 kit is 2% sodium dodecylsulfate (SDS) and 1 M βME, and 2.5μl is used in a reaction volume of around 50μl giving a final reaction concentration of 0.1% SDS, 50mM bME. Denaturation Solution and 5x Incubation Buffer are added to the glycoprotein substrate and the mixture is heated to 100°C for 5 minutes. After cooling, 2.5μl of a Detergent Solution (15% NP-40, final reaction concentration 0.75%) is added to counteract the inhibitory effect of SDS on PNGase F. This is followed by the addition of N-Glycanase, Sialidase A (GK80040) and O-Glycanase (GK80090), with incubation for 3 hours at 37°C.