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] FrankMaley and Robert B. Trimble and Anthony L. Tarentino and Thomas H. Plummer Jr. Characterizationof glycoproteins and their associated oligosaccharides through the use ofendoglycosidases[J]. Analytical Biochemistry, 1989.. [2] B, Ling Hua A, et al. Highly efficient productionof peptides: N -glycosidase F for N -glycomics analysis[J]. Protein Expressionand Purification, 2014, 97(5):17-22. |
Background
Peptide:N-glycosidase F (PNGase F) is an asparagine amidase produced by Flavobacteriummeningosept-icum that serves as a useful tool in the research on proteinN-glycosylation. The cleavage site of PNGase F is the amide bond betweenN-acetylglucosamine (GlcNAc) and aspartate residues on the medial side of theglycoprotein, and converts aspartyl to aspartic acid on the enzymolysisprotein. This productoverexpressed in E.coliand used for complete deglycosylation of antibodies and their associatedproteins
Components
StorageSolution : 40U/ul PNGaseF、20 mM Tris-HCl、50 mM NaCl, 5 mM EDTA(pH 7.5 @ 25°C)
10*NP-40: 10% NP-40 inMilliQ-H2O
10*Denaturing Buffer: 5% SDS、400 mM DTT
10*Reaction Buffer: 500mMTris-HCl (pH 7.5 @ 25°C)
Protocol
1. 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
2. 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
