M marker
Lane 1 Rnase B 10μg
Lane 2 Rnase B 10μg+0.25U PNGF
Lane 3 Rnase B 10μg+0.5U PNGF
Lane
4 Rnase B 10μg+1U PNGF
PNGase F(Glycerol-free)
PNGase F(Glycerol-free)
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$200 USD
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Product Details
Product Details
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
One unit of enzyme activity refers to the amount of enzyme required to remove more than 95% of carbohydrate from 10μg denatured RNaseB at 37℃ for 1 hour in a 10μL reaction system.
Picture
Picture
Bioactivity
RP-HPLC
