Peptide-N-Glycosidase F (PNGase F)
Peptide-N-Glycosidase F (PNGase F)
Cat. No.: SB-G3468-25000U
Size: 25000U
Description
Peptide-N-Glycosidase F , referred to as PNGase F, is derived from glycosidase F of Chryseobacterium miricola, recombinantly expressed in Escherichia coli, and can cleave high mannose, hybrid and complex oligosaccharide glycoproteins linked by asparagine. The cleavage site of PNGase F is the amide bond between N-acetylglucosamine (GlcNAc) and asparagine residues on the side of the glycoprotein, and at the same time converts aspartyl on the protein after enzymatic hydrolysis to aspartic acid. Mainly used to remove N-glycosylation of proteins.
Source: derived from Chryseobacterium miricola, recombinantly expressed in Escherichia coli;
Definition of enzyme activity: at 37°C, the amount of enzyme required to remove more than 95% of carbohydrates in 10 μg of denatured RNase B for 1 hour is defined as an enzyme activity unit;
Purity and concentration: SDS-PAGE detection purity > 95%; endogenous nucleic acid residue < 1 pg/μL (qPCR detection); 500 U/μL;
Inactivation or inhibition: Heat at 75°C for 10 minutes to inactivate;
Enzyme storage buffer: 20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50% Glycerol, pH 7.5;
10х Denaturing Buffer: 5% SDS, 400 mM DTT;
10х Native Buffer: 400 mM sodium Phosphate, pH 7.5;
Figure 1. Deglycosylation effect of substrate RNase B treated with PNGase F. After the substrate RNase B was denatured, 10 μg was added to different enzyme amounts of PNGase F (0, 10, 1, 0.5, 0.1 U), incubated at 37°C for 1 h, and detected by PAGE electrophoresis.
Storage and transportation
Transport in wet ice packs; store at -20°C, valid for 12 months.
Product Contents
| Component Number | Component | G3468 |
| G3468-1 | Peptide-N-Glycosidase F (PNGase F) | 50 μL |
| G3468-2 | 10х Denaturing Buffer | 1mL |
| G3468-3 | 10х Native Buffer | 1mL |
| Product Manual | 1 copy | |
Steps
1. Protein deglycosylation under denaturing conditions:
Add 1 μL 10х Denaturing Buffer and dH2O to the 1-20 μg protein sample to the 10 μL reaction system, and incubate the system at 100°C for 10 min. Then add 2 μL 10х Native Buffer, 2 μL 10% NP-40, 1 μL PNGase F and appropriate amount of dH2O to the 20 μL reaction system, and incubate at 37°C for 1 h. (Note: The activity of PNGase F will be inhibited by SDS, so NP-40 must be added under denaturing conditions)
2. Protein deglycosylation under non-denaturing conditions:
Add 2 μL 10х Native Buffer, 2-5 μL PNGase F and appropriate amount of dH2O to 20 μL reaction system in 1-20 μg protein sample , and incubate at 37°C for 4-24 h. (Protein deglycosylation under non-denaturing conditions requires longer reaction times or more enzymes)
Precautions
1. The activity of PNGase F will be inhibited by SDS, so NP-40 must be added under denaturing conditions.
2. The easiest way to assess the degree of protein deglycosylation is by SDS-PAGE gel electrophoresis.
3. All enzymes should be stored in an ice box and stored at -20°C immediately after use.
4. For your safety and health, please wear a lab coat and disposable gloves for operation.
