Home Protocols Reaction Conditions for Endo D (P0742)

Reaction Conditions for Endo D (P0742)

Protocol

Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows:

  1. Combine 10–20 μg of glycoprotein, 1 μl of 10X DTT and H2O (if necessary) to make a 10 μl total reaction volume.
  2. Denature the glycoprotein by heating the reaction at 55°C for 10 minutes.
  3. Make a total reaction volume of 20 μl by adding 2 μl 10X GlycoBuffer 2, H2O and 1–5 μl Endo D .
  4. Incubate the reaction at 37°C for 1 hour.
Note: To deglycosylate various glycoproteins under native conditions, longer incubation times as well as more enzyme may be required. Reactions may be scaled-up linearly to accommodate larger reaction volumes.