New England Biolabs, P8113S, α-Lytic Protease

α-Lytic Protease (aLP) cleaves after Threonine (T), Alanine (A), Serine (S) and Valine (V) residues. Its specificity makes it an orthogonal and alternative protease to others commonly used in proteomics applications, including trypsin and chymotrypsin. Related Categories Proteases,, Proteome Analysis Applications Protein Analysis Tools,, Protein Digestion,, Proteomics Specification Storage Buffer 10 mM sodium acetate pH 5 @ 25°C Heat Inactivation 95°C Molecular Weight Apparent: 19860 daltons FAQ Q: What reaction buffers are recommended for use with α-Lytic Protease? A: α-Lytic Protease is active in a variety of buffers including ammonium bicarbonate, Tris-HCl, and HEPES. The optimal pH range for α-Lytic Protease digestion is pH 7.5 - 8.5. For downstream mass spectrometric analysis, ammonium bicarbonate buffer is recommended. Q: Can I perform my digest with α-Lytic Protease at temperatures other than 37°C? A: Yes, α-Lytic Protease is active between 4°C - 50°C. Extended incubation times may be necessary for digests at lower temperatures. The recommended digestion time at 37°C ranges from 1 to 18 hours depending upon the substrate and desired extent of digestion. Q: Why should I use a-Lytic Protease instead of Trypsin A: α-Lytic Protease may better digest proteins containing large hydrophobic regions or membrane-associated proteins compared to Trypsin, thereby providing enhanced sequence coverage and allowing for identification of novel post-translational modifications. Q: Is there an advantage to treating my sample with both α-Lytic Protease and Trypsin? A: Yes. Site-specific cleavage after lysine and arginine residues by trypsin creates peptides that are positively charged but may be too large for mass spectrometric detection. Performing a double digest of the substrate with Trypsin and α-Lytic Protease creates smaller peptides, thereby increasing both sequence coverage and the likelihood of identifying novel post-translational modifications. Q: What reagents inhibit α-Lytic Protease activity? A: α-Lytic Protease 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 Q: What reagents stimulate α-Lytic Protease activity? A: α-Lytic Protease activity is stimulated in up to 0.1% sodium deoxycholate. Q: Can α-Lytic Protease be heat-inactivated? A: Yes. α-Lytic Protease can be heat-inactivated by incubating at 95°C for 10 minutes. Incubation at 70°C abolishes only ~50% of enzymatic activity.