PNGase F(Glycerol-free),specification,price,image-Bio-Equip in China

Product Detail
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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.