New England Biolabs, P8109S, Endoproteinase LysC

Endoproteinase LysC is a serine Endoproteinase which cleaves peptide bonds at the carboxyl side of lysine. LysC is a sequencing grade enzyme and is suitable for proteomics and glycobiology applications. Related Categories Proteases,, Proteome Analysis Applications Glycomics and glycoproteomics,, Biotherapeutics and Antibody Analysis,, Protein Digestion, Specification Usage Concentration 100 ng/μl Molecular Weight Apparent: 30000 daltons FAQ Q: What buffer conditions can Endoproteinase LysC be used in? Are there any special applications for which LysC is especially good? A: LysC is active in a wide pH range and can also function under some denaturing e.g., as high as 5-8 M urea. For this reason, the enzyme is often used on difficult to digest proteins or protein mixtures. Typically, buffer pH range between 7-9 is used. Endoproteinase LysC can be used in acidic buffer conditions including, 20 mM sodium acetate pH 4.5-6.5. Q: How much Endoproteinase LysC do I really need to digest my protein? A: We recommend LysC : substrate ratio within 1:50-100. However, we have observed significant digestion products using as low as 1:250. A database search revealed 65% sequence coverage for the latter and > 80% coverage for the former. Q: Can Endoproteinase LysC used in a double digest with Trypsin-ultra? A: Trypsin-LysC co-digests have been found to improve protein coverage in proteomics studies especially among low abundance proteins. Many different conditions have been used successfully. Please refer to the references below for more detailed discussions. A good standard Trypsin-LysC digest is to set up a LysC digest in 8M urea buffer for 4hrs at 37 °C, then dilute to 1.5 M urea for a trypsin digest for 4 hrs at 37 °C. Both digests should be 1:20-50 protease to susbtrate protein. References: Giansanti, P. et al 2016 “Six alternative proteases for mass-spectrometry based proteomics beyond trypsin” Natur Protoc. 11, 993-1006. Swaney, D. L. et al 2010 “The value of using multiple proteases for large-scale mass spectrometry-based proteomics” J. Proteome Res. 9, 1323-1329. Wang, Y. et al 2016 “Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid-chromatography-mass spectrometry” Anal. Biochem. 495, 21-28. Q: Can Endoproteinase LysC be used in acidic buffer conditions? A: Yes, Endoproteinase LysC can be used in acidic buffer conditions. Robust cleavage yielding the products has been observed in the following buffers: 20 mM sodium acetate pH 4.5 20 mM sodium acetate pH 5.5 20 mM sodium acetate pH 6.0 20 mM sodium acetate pH 6.5 Digests can also be run in Tris buffer between a pH range of 7.0 - 9.1.