α-Lytic Protease Typical Reaction Protocol (NEB #P8113)
For peptide or protein digestion, a ratio between 1:20 to 1:100 (w/w) of enzyme to substrate is recommended. Reactions may be scaled-up linearly to accommodate larger amounts of substrate and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:
- Combine 2 μg substrate and 50 mM ammonium bicarbonate pH 8.5 to make a 10 μl total reaction volume.
- Add α-Lytic Protease in a 1:20 – 1:100 (α-Lytic Protease : substrate) ratio by mass.
*Note: For example, add 20-100ng α-Lytic Protease to 2 μg substrate protein
- Incubate at 37°C for 1 hour.
*Note: longer incubations (18 hours) or more enzyme may be required, especially at lower temperatures, for complete digestion.
- α-Lytic Protease is active in a variety of buffers including ammonium bicarbonate, Tris-HCl, and HEPES.
- Enzyme activity is stimulated in up to 0.1% sodium deoxycholate.
- Enzyme activity is inhibited by 1.0% sodium deoxycholate (∼60% active); 0.1% SDS (~50 active); 1% SDS (40% active); 1 M guanidine hydrochloride (∼20% active); 4 M guanidine hydrochloride (no activity); Serine protease inhibitors, such as PMSF (no activity).
- α-Lytic Protease is active over a wide temperature range from 4ºC – 50ºC, with optimal activity at 37ºC.