Quality control assay: Denatured RNaseB and human monoclonal antibody were used as substrates to test PNGase F activity. (Figure above)
Stability: PNGase F retains >60% activity after left at room temperature for over 72 hours. Long term storage at - 20 C or below
Purity: >95% by SDS-PAGE gel
Unit definition: One unit is defined as the amount of enzyme required to removed >95% of the glycans from 10 ug of denatured RNase B in 1 hour at 37 C.
Concentration: 50,000 units/mL
1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
Protocol for denaturing conditions
1. Denature 20 ug of glycoprotein in 0.5% SDS, 50mM DTT and 50mM Tris buffer pH8 by heating at 95 C for 5 minutes.
2. Then add NP-40 or Triton X-100 to sample (1% final concentration).
3. Add 10 uL PNGase per 20 ug of glycoprotein
4. Incubate at 37 C for 1 hour.
Protocol for non-denaturing conditions
1. Add 50 uL PNGase per 20 ug of glycoprotein in 50mM Tris buffer pH8
2. Incubate at 37 C for 18 hours.
1. Recommended substrate concentration for reaction is 0.1-1 mg/mL
2. NP-40 or Triton X-100 prevents SDS inhibition of PNGase F activity.
3. PNGase F does not cleave N-glycans containing core a1-3 fucose.
|Product Name：||PNGase F|
|Formulation：||20 mM Tris pH8, 50% glycerol.|
|Purity：||>95% by SDS-PAGE gel|
|Storage：||Long term storage at -20 C or below|