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  • Reaction Conditions for Remove-iT® Endo D (P0742)


    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 Remove-iT 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.