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Description
PNGase F(Glycerol-free)Product Specification Species Elizabethkingia miricola Synonyms Peptide N(4) (N acetyl beta D glucosaminyl)asparagine amidase F, Peptide N Glycosidase F, PNGase F Expression System E. coli Molecular Weight 36kDa (Reducing) Purity 95% by SDS PAGE and HPLC Conjugation Unconjugated Tag His Tag Physical Appearance Liquid Storage Buffer 20 mM Tris HCl50 mM NaCl5 mM EDTApH 7. 5 @ 25C Stability & Storage Store at 25 ~ 15 for 2 years Reference [1] Frank Maley
Product Specification
| Species | Elizabethkingia miricola |
| Synonyms | Peptide-N(4)-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F, Peptide N-Glycosidase F, PNGase F |
| Expression System | E.coli |
| Molecular Weight | 36kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE and HPLC |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 20 mM Tris-HCl、50 mM NaCl、5 mM EDTA（pH 7.5 @ 25°C） |
| Stability & Storage |
Store at -25 ~ -15℃
for 2 years
|
| Reference |
[1] Frank Maley and Robert B. Trimble and Anthony L. Tarentino and Thomas H. Plummer Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases[J]. Analytical Biochemistry, 1989..
[2] B, Ling Hua A, et al. Highly efficient production
of peptides: N -glycosidase F for N -glycomics analysis[J]. Protein Expression
and Purification, 2014, 97(5):17-22.
|
Background
Peptide:
N-glycosidase F (PNGase F) is an asparagine amidase produced by Flavobacterium
meningosept-icum that serves as a useful tool in the research on protein
N-glycosylation. The cleavage site of PNGase F is the amide bond between
N-acetylglucosamine (GlcNAc) and aspartate residues on the medial side of the
glycoprotein, and converts aspartyl to aspartic acid on the enzymolysis
protein. This productoverexpressed in E.coli
and used for complete deglycosylation of antibodies and their associated
proteins
Components
Storage Solution : 40U/ul PNGase F、20 mM Tris-HCl、50 mM NaCl, 5 mM EDTA (pH 7.5 @ 25°C)
10*NP-40: 10% NP-40 in MilliQ-H2O
10*Denaturing Buffer: 5% SDS、400 mM DTT
10*Reaction Buffer: 500mM
Tris-HCl (pH 7.5 @ 25°C)
Protocol
一. Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 1 µl of Denaturing Buffer (10X) and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denaturation is terminated by heating to 100℃ for 10-20min and cooling to room temperature.
3. Make a total reaction volume of 20 µl by adding 2 µl Reaction Buffer (10×), 2 µl 10% NP-40 and 6 µl H2O.
4. Add 1 µl PNGase F, mix gently.
5. Enzymatic digestion at 37℃ for 1h
6. Analyze by the method of SDS-PAGE
二. Non-Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 2 µl of Reaction Buffer (10×) and H2O (if necessary) to make a 20 µl total reaction volume.
2. Add 2-5 µl PNGase F, mix gently.
3. Enzymatic digestion at 37°C for 4 - 24 hours.
4. Analyze by the method of SDS-PAGE
Unit Definition
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