Home Protocols Reaction Protocols for Protein Deglycosylation Mix (P6039)

Reaction Protocols for Protein Deglycosylation Mix (P6039)

Introduction

The quantity of enzyme recommended is sufficient for the deglycosylation of 100 µg of a glycoprotein. Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:

Protocol

  1. Denaturing Reaction Conditions:

    1. Dissolve 100 µg of glycoprotein into 18 µl H2O.
    2. Add 2 µl of 10X Glycoprotein Denaturing Buffer to make a 20 µl total reaction volume.
    3. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
    4. Chill denatured glycoprotein on ice and centrifuge 10 seconds.
    5. To the denatured glycoprotein reaction add 5 µl 10X GlycoBuffer 2, 5 µl 10% NP-40, and 15 µl H2O. 
      PNGase F and O-Glycosidase are inhibited by SDS, therefore it is essential to have NP-40 in the reaction mixture under denaturing conditions. Failure to not include NP-40 into the denaturing protocol may result in loss of activity of some enzymes.
    6. Add 5 µl Deglycosylation Enzyme Cocktail, mix gently.
    7. Incubate reaction at 37°C for 4 hours.
    8. Analyze by method of choice 
      Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
  2. Non-Denaturing Reaction Conditions: 
    When deglycosylating a native glycoprotein it is recommended that an aliquot of the glycoprotein is subjected to the denaturing protocol to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.
    1. Dissolve 100 µg of glycoprotein into 40 µl H2O.
    2. To the native glycoprotein add 5 µl 10X GlycoBuffer 2.
    3. Add 5 µl Deglycosylation Enzyme Cocktail, mix gently.
    4. Incubate reaction at 37°C for 4 hours.
      Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
    5. Analyze by method of choice.
      Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.