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Endoglycosidases >
PNGase F (Glycerol Free) |
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Prices are in US dollars and valid only for US orders.
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- Supplied with 10X G7 Reaction Buffer, 10X Glycoprotein Denaturation buffer, and 10% NP-40
Description:
Peptide: N-Glycosidase F, also known as PNGase F, is an amidase and supplied glycerol free for optimal performance in HPLC intensive methods. PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins (1)
PNGase F hydrolyzes nearly all types of N-glycan chains from glycopeptides/proteins. [x = H or sugar(s)]
Source:
PNGase F is purified from Flavobacterium meningosepticum (2).
Applications:- Removal of carbohydrate residues from proteins
- Preferred formulation for HPLC intensive methods
Reagents Supplied:
G7 Reaction Buffer (10X)
Glycoprotein Denaturing Buffer[5% SDS, 0.4M DTT] (10X)
NP-40 (10%)
Enzyme Properties
Molecular Weight:
Apparent: 36,000 daltons
Specific Activity:
1,800,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X G7 Reaction Buffer
Supplemented with 1 % NP-40
Incubate at
37°C.
1X G7 Reaction Buffer:
50 mM sodium phosphate
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 µl (65 NEB units = 1 IUB milliunit).
Unit Definition Assay:
10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and G7 Reaction Buffer, two-fold dilutions of PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.
Concentration:
500,000 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
5 mM Na2EDTA
pH 7.5 @ 25°C
Storage Temperature:
4°C
Store at 4°C, do not freeze.
Notes
Usage notes:- Since PNGase F activity is inhibited by SDS, it is essential to have NP-40 present in the reaction mixture. Why this non-ionic detergent counteracts the SDS inhibition is unknown at present.
- To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
- PNGase F will not cleave N-linked glycans containing core α1-3 Fucose.
- Repeated freeze thaw cycles degrade enzyme activity over time.
- Activity at different temperatures (measured after a 1 hour incubation of glycosidase and denatured RNase B at the given temperature): 37°C - 100%; 30°C - 100%; 23°C - 65%; 17°C - 40% and 3°C - 0%.
- Typical reaction conditions: Please see FAQs.
FAQs
- I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
- What is the difference between PNGase F and Endo H?
- How much PNGase F should I use to remove my carbohydrate under native conditions?
- How do I inhibit PNGase F?
- What is a good endoglycosidase substrate?
- Does PNGase F work in Urea?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- What are the typical reaction conditions for PNGase F (Glycerol Free)?
- Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?
- What are Glycosidases and their uses?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
No contaminating exoglycosidase or Endoglycosidase F2 or F3 activity could be detected. < 0.01% of Endoglycosidase F1 could be detected. No contaminating proteolytic activity could be detected.
Glycosidase Assay:
5,000 units of PNGase F were incubated with 0.1 mM of fluorescently-labeled oligosaccharides and glycopeptides, in a 10 µl reaction for 20 hours at 37°C. The reaction products were analyzed by TLC for digestion of substrate.
No other glycosidase activities were detected (nd) with the following substrates:
β-N-Acetyl-glucosaminidase: GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC (nd)
α-Fucosidase: Fucα1-2Galβ1-4Glc-AMC Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC (nd)
β-Galactosidase: Galβ1-3GlcNAcβ1-4Galβ1-4Glc-AMC (nd)
α-Galactosidase: Galα1-3Galβ1-4Galα1-3Gal-AMC (nd)
α-Neuraminidase: Neu5Acα2-3Galβ1-3GlcNAcβ1-3 Galβ1-4Glc-AMC (nd)
α-Mannosidase: Manα1-3Manβ1-4GlcNAc-AMC Manα1-6Manα1-6(Manα1-3)Man-AMC (nd)
β-Glucosidase: Glcβ1-4Glcβ1-4Glc-AMC (nd)
β-Xylosidase: Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC (nd)
β-Mannosidase: Manβ1-4Manβ1-4Man-AMC (nd)
Endo F1, F2, H: Dansylated invertase high mannose (nd)
Endo F2, F3: Dansylated fibrinogen biantennary (nd)
Protease Assay:
After incubation of 10,000 units of PNGase F with 0.2 nmol of a standardized mixture of proteins, for 20 hours at 37°C, no proteolytic activity could be detected by SDS-PAGE.
References
- Maley, F. et al. (1989) Anal. Biochem., 180, 195-204.
- Plummer, T.H. Jr. and Tarentino, A.L. (1991) Glycobiology, 1, 257-263.
Companion Products
Endo H
Endo Hf
Endoglycosidase Reaction Buffer Pack
PNGase F
RNase B
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