| Source : |
E.coli |
| Tag : |
Non |
| Description : |
It can selectively hydrolyze all N-glycan chains linked to Asn in proteins, except those containing α1-3 fucosidic bonds. The cleavage site is the glycosidic bond between Asn and the innermost GlcNAc. This causes the glycoprotein to release the Asn-linked sugar molecules and converts Asn to Asp. The cleaved glycan types include high-mannose, hybrid, and complex oligosaccharides. |
| Form : |
Lyophilized |
| Purity : |
≥ 95% by SDS-PAGE |
| Advantages : |
1. High purity: It is free from contamination by other proteases and has no other endo-and exo-glycosidase activities, with a purity of ≥ 95%;
2. High stability: Each batch of PNGase F undergoes strict quality control to ensure batch-to-batch stability of the product;
3. High specific activity: It can effectively and completely release N-linked glycans;
4. Compatible with HPLC/ MS: The lyophilized form of PNGase F does not contain glycerol, which helps to achieve optimal results in HPLC and MS analyses;
5. This product can cleave glycoproteins under denaturing and non-denaturing conditions. |
| Protein Content : |
≥ 20 KU |
| Unit definition : |
One unit is defined as the amount of the enzyme required to remove more than 95% of the carbohydrates from 10 μg of denatured RNase B in a 10 μL reaction system at 37 centigrade for 1 hour. |
| Usage : |
1. Protein Reconstitution
Centrifuge the centrifuge tube containing lyophilized PNGase F at 100-200 g for 2 minutes. Carefully open the cap and add pure water to achieve a final concentration of 500 U/μL. Invert the tube up and down to mix thoroughly as much as possible. After mixing, centrifuge at 100-200 g for 2 minutes. The reconstituted PNGase F should be stored at-20 centigrade for 6 months, and users should try to avoid repeated freeze-thaw cycles.
2. Denaturing enzymatic digestion
Dissolve 1-20 μg of glycoprotein in deionized water. Add 1 μL of 10× glycoprotein denaturation buffer and adjust the volume to 10 μL with deionized water.
Incubate at 100 centigrade for 10 min.
Transfer to ice and centrifuge for 10 s.
Add 2 μL of 10× PNGase F digestion reaction buffer, 2 μL of 10% NP-40, and 5 μL of deionized water. Mix well.
Add 1 μL of the reconstituted PNGase F.
Incubate at 37 centigrade for 60 min.
Analyze the effect of enzymatic digestion.
3. Non-denaturing enzymatic digestion
Dissolve 1-20 μg of glycoprotein in deionized water. Add 1 μL of 10× PNGase F digestion reaction buffer and 2-4 μL of the reconstituted PNGase F, then adjust the volume to 10 μL with deionized water.
Incubate at 37 centigrade for 2 hours to overnight.
Analyze the effect of enzymatic digestion. |
| Note : |
1. Denaturing enzymatic digestion has a relatively high efficiency and should be attempted first. If denaturing enzymatic digestion cannot be carried out (for example, precipitation occurs during the denaturation process), non-denaturing enzymatic digestion can be tried. However, the digestion efficiency may decrease. It is recommended to increase the amount of enzyme and extend the digestion time;
2. After reconstitution, try to avoid repeated freeze-thaw cycles for this product;
3. Please wear lab coat and disposable gloves when using;
4. This product should not be used directly for clinical diagnosis and treatment. |
| Storage : |
The PNGase F, Lyophilized should be stored at 2-8 centigrade. The 10× glycoprotein denaturation buffer, 10× PNGase F digestion reaction buffer and 10% NP-40 should be stored at-20 centigrade and can be preserved for at least 12 months. |
| Shipping : |
Dry ice or ice packs |
| Reconstitution : |
Ultrapure water |
