| Description : |
The enzyme hydrolyze the N-terminal of the asparagine (Asn) residue side chain to release asparagine-linked oligosaccharides from glycoproteins and glycopeptides. The oligosaccharides can be high mannose, hybrid, or complex type. |
| Form : |
rPNGase F in 20 mM Tris-HCl (pH 7.5 at 25 centigrade), 50 mM NaCl, and 5 mM EDTA solution |
| Bio-activity : |
40,000 units/min/mg |
| Molecular Mass : |
35 kDa |
| Purity : |
> 99.0 % rPNGase F peak area analyzed by HPLC at 280 nm. |
| Storage : |
Store at 4 centigrade. |
| Concentration : |
10000 U/mL (1U=65 NEB units) |
| CAS No. : |
83534-39-8 |
| Shelf life : |
24 months at 4 centigrade |
| Stability : |
rPNGase F is maximally active in the pH range of 6-10, best at pH 8.6. |
| Usage Notes : |
In-Solution Protein Digestion Protocol:
1)Denaturation enzyme digestion:
1. For maximum activity, resuspend the user's samples (eg. glycoprotein) with 50 mM ammonium bicarbonate buffer (pH 7.8).
2. Dissolve 20 μg samples in 50 mM ammonium bicarbonate (pH 7.8) to a final volume of 18 μL.
3. Add 2 μL rPNGase F to the solution in step 2 Mix well.
2)Native enzyme digestion:
1. For maximum activity, resuspend the user's samples (eg. glycoprotein) with 50 mM ammonium bicarbonate buffer (pH 7.8).
2. Dissolve 50 μg samples in 50 mM ammonium bicarbonate (pH 7.8) to a final volume of 50 μL.
3. Add 1 μL rPNGase F to the solution in step 2 Mix well and incubate at 37 centigrade for 1-2 hours. |
| Unit Definition : |
One unit catalyzes the release of N-linked oligosaccharides from 1 nanomole of denatured Ribonuclease B in 1 min at 37 centigrade at pH 7.5 monitored by LC-MS. |
| LC-MS/MS Analysis : |
Denaturated RNase B was digested with rPNGase F at 37 centigrade for 60 minutes, and LCMS analysis showed that RNase B removed more than 98% of N-glycans. |
