Home Protocols Typical Reaction Conditions (P0708)

Typical Reaction Conditions (P0708)

Typical reaction conditions are as follows:
  1. Combine 1–20 μg of glycoprotein, 1 μl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 μl total reaction volume.
  2. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
  3. Make a total reaction volume of 20 μl by adding 2 μl 10X G7 Reaction Buffer, 2 μl 10% NP40, H2O and 1–2 μl PNGase F, Recombinant.
  4. Incubate reaction at 37°C for 1 hour.
Notes
  • If using P0708, we recommend limiting PNGase F to 1/10 (or less) of the total reaction volume to keep final glycerol concentration equal to (or less than) 5%.
  • Reaction may be scaled up linearly to accommodate large amounts of PNGase F and larger reaction volumes.
  • Several exo- and endo-glycosidases can be used together in one digest.  For buffer recommendations, please reference the Glycosidases Double Digest Chart .
  • For a more detailed characterization of several glycosidase enzymes, please reference the Detailed Characterization of Several Glycosidase Enzymes page.
  • For unit conversion between different suppliers, please reference the Glycobiology Unit Conversion Chart page.