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

Reference:

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.

Note:

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.