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:
- 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.
- Denature glycoprotein by heating reaction at 95°C for 10 minutes.
- Cool the reaction to room temperature.
- Add 3 μl of 500 mM iodoacetamide.
- Incubate at 25°C (room temperature) for 30 minutes.
- Transfer reaction into a centrifugal filter device, such as Nanosep® Centrifugal Device with Omega™ Membrane 10K (Pall Life Sciences catalog #OD010C33).
- Centrifuge filter device at 12000 x g for 4 minutes, discard flow through.
- Add 400 μl of Wash Buffer: 20 mM Tris-HCl, pH 8.0.
- Centrifuge filter device at 12000 x g for 4 minutes, discard buffer. Repeat once.
- Add 50 μL of 20 mM Tris-HCl, pH 8.0.
- Add 1 μL of O-Glycoprotease, mix gently.
- 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).
- Centrifuge at 12000 x g for 4 minutes and collect supernatant.
- 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.