Home Protocols Reaction Protocols for Protein Deglycosylation Mix II (P6044)

Reaction Protocols for Protein Deglycosylation Mix II (P6044)

Introduction

The quantity of enzyme recommended is sufficient for the deglycosylation of 100 µg of a glycoprotein. Reactions may be scaled-up or down linearly to accommodate other amounts of glycoprotein or different reaction volumes. Optimal incubation times may vary for particular substrates. For most glycoproteins, deglycosylation is more extensive under denaturing conditions. Both protocols are compatible with downstream mass spectrometry analysis.

Protocol

1.    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. Combine up to 100 µg of glycoprotein and water to a volume of 40 μL
  2. To the native glycoprotein add 5 µl 10X Deglycosylation Mix Buffer 1.
  3. Add 5 µl Protein Deglycosylation Mix II, mix gently.
  4. Incubate reaction at 25°C (room temperature) for 30 minutes.
  5. Transfer reaction to 37°C, incubate for 16 hours.
  6. Analyze by method of choice.

    Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.

2.    Denaturing Reaction Conditions:

  1. Combine up to 100 µg of glycoprotein and water to a volume of 40 μL
  2. Add 5 µl of Deglycosylation Mix Buffer 2.
  3. Incubate at 75°C for 10 minutes, cool down.
  4. Add 5 µl Protein Deglycosylation Mix II, mix gently.
  5. Incubate reaction at 25°C (room temperature) for 30 minutes.
  6. Transfer reaction to 37°C, incubate for 1 hour

    Note: most glycoproteins will be deglycosylated after 1 hour at 37°C. However, some complex glycoproteins may require a longer, 16 hour incubation.

  7. Analyze by method of choice

    Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels. To prepare samples for MS, we recommend a buffer exchange by dialysis or microcentrifugation. Please reference protocol “Glycoproteomics: Buffer Exchange Protocols (P0710) ”