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  • FAQ: Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?

    When a protein is denatured it is more susceptible to cleavage by proteases. For this reason a protease cocktail containing the following can be used in a PNGase F reaction protocol:
    Aprotinin (10 μg/ml final concentration)
    Benzamidine (1mM final concentration)
    Pepstatin (10 μg/ml final concentration)
    Leupeptin (1μM final concentration)
    EGTA (1 mM final concentration)
    EDTA (1 mM final concentration)
    PMSF (1 mM final concentration)
    Make a 1000X concentrated stock of each inhibitor in water; except Pepstatin which should be dissolved in methanol.
    Note: PMSF has the ability to modify basic residues on glycoprotein substrates.