sin with Lys-C? Get Better Data from Poorly Purified Protein Extracts The number of missed cleavage sites may be even higher if the protein is poorly purified or contains protease-inhibiting contaminants. For example, guanidine hydrochloride, a common agent for solubilizing and denaturing proteins, inhibits trypsin even at low concentrations. The Trypsin/Lys-C Mix is not inhibited by these reagents, thus enabling the generation of data from poor quality material. Learn More in the Trypsin/Lys-C Webinar Percent Missed Cleavages 57% 5.8% Trypsin Trypsin/Lys-C Mix Trypsin Trypsin/Lys-C Mix Tenfold decrease in missed cleavages. 40% increase in unique peptides. Trypsin Trypsin/Lys-C Mix 17% increase in identified proteins. Digestion of proteins resistant to proteolysis. Trypsin/Lys-C Mix improves overall digestion due to Lys-C tolerance to denaturing conditions. Unique Peptides 1490 2092 415 Identified Proteins 484 Overcome Digestion Limitations without Inhibiting Trypsin Activity Tightly folded proteins represent another challenge for trypsin. Theoretically, proteolytically resistant proteins can be digested under strong denaturing conditions; however, denaturing conditions inhibit trypsin. Using the Trypsin/Lys-C Mix, proteolysis is performed in two steps. The first step uses a strong protein denaturing agent such as urea, which allows Lys-C to cleave previously inaccessible sites. Lys-C digests proteins into relatively large fragments, while trypsin activity is inhibited. In the second step, the digestion mixture is diluted to reduce urea concentration. This reactivates trypsin and allows complete proteolysis. kDa M1 23 4 98 64 50 36 16 6 Myoglobin Myoglobin peptides Trypsin/Lys-C Mix Order Digestion of Proteolytic Resistant Protein Using Trypsin/Lys-C Mix. Horse myoglobin was digested as follows: Lane 1, no-protease control; lane 2, trypsinonly digestion under nondenaturing conditions; lane 3, digestion with Lys-C in 8M urea; lane 4, two-step digestion with Trypsin/Lys-C Mix. enews | Fall 2013 Pagina 10
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