O-Glycoprotease On-filter Cleavage Denaturing Reaction Protocol (NEB #P0761)

Optimal enzyme concentration and incubation times may vary for a particular substrate. Reactions may be scaled-up linearly to accommodate larger amounts of substrate and larger reaction volumes. Typical reaction conditions are as follows:

  1. Combine 10 µg of glycoprotein in 20 mM Tris-HCl pH 8.0, 40 mM DTT, 0.1% SDS to a total reaction volume of 50 μL.

  2. Denature glycoprotein by heating reaction at 95°C for 10 minutes.

  3. Cool the reaction to room temperature.

  4. Add 3 μl of 500 mM iodoacetamide.

  5. Incubate at 25°C (room temperature) for 30 minutes.

  6. Transfer reaction into a centrifugal filter device, such as Nanosep® Centrifugal Device with Omega™ Membrane 10K (Pall Life Sciences catalog #OD010C33).

  7. Centrifuge filter device at 12000 x g for 4 minutes, discard flow through.

  8. Add 400 μl of Wash Buffer: 20 mM Tris-HCl, pH 8.0.

  9. Centrifuge filter device at 12000 x g for 4 minutes, discard buffer. Repeat once.

  10. Add 50 μL of 20 mM Tris-HCl, pH 8.0.

  11. Add 1 μL of O-Glycoprotease, mix gently. 

  12. Incubate the reaction on the column at 37˚C for 2 hours.


    Note - Some substrates may require extended incubation periods (up to 18 hours at 37°C).

  13. Centrifuge at 12000 x g for 4 minutes and collect supernatant.

Notes:

  • Recommended substrate to enzyme ratio is 10 µg substrate to 1 µL enzyme.

  • The optimal pH range for O-Glycoprotease is pH 7.0-8.0. Enzyme activity decreases significantly at pH values below 7.0 and above pH 8.0.

  • Reactions can be filtered or used immediately for analysis by MS or MS/MS.

  • Reactions can be stored frozen at -20°C for later use.