In many proteome studies, sample analysis is performed directly from the liquid phase. This method avoids many of the time consuming steps associated with in-gel digestion. As with in-gel digestions, trypsin is the enzyme of choice. Trypsin cleaves at C-terminal of lysine (K) and arginine (R) amino acids resulting in a N-terminal amine group that can accept a proton and a basic side chain residue of the K/R that will also take up a proton. These charged particles can be easily ionized when analyzed by mass spectrometry.
Immobilized Trypsin provides a fast and convenient method for digesting a range of concentrations of purified protein, or complex protein mixtures. It is based on immobilized trypsin on cellulose particles. The appropriate amount of particles is then packed in the provided spin column. After three brief washing/equilibration steps, a sample consisting of 20-500µg of protein is added to the column. This enables researchers to digest a variety of protein samples without requiring additional concentration steps or optimization.
After a 30-minute digestion, peptides are easily separated from the Immobilized Trypsin by brief centrifugation as they flow through the spin column into the collection tube. Immobilized Trypsin is easily removed from the peptide solution because the trypsin does not pass though the column frit.
The resulting peptides can be analyzed via mass spectrometry.
Immobilized Trypsin Technical Manual #TM077
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Sr. Product Manager for Protein Analysis products.
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Gary Kobshttps://www.promegaconnections.com/author/gkobs/
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Gary Kobshttps://www.promegaconnections.com/author/gkobs/
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Gary Kobshttps://www.promegaconnections.com/author/gkobs/
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Gary Kobshttps://www.promegaconnections.com/author/gkobs/
thanks for sharing this one!