Immobilized Trypsin Protein Digestion in as little as
Many protein digests are performed in solution to avoid the time-consuming steps associated with in-gel digestion. Trypsin is the enzyme of choice for creating small peptide fragments for mass spectrometry analysis. Trypsin cleaves at the C-terminus of lysine (K) and arginine (R) amino acids, resulting in an Nterminal amine group. The basic side chain residues of K, R and the N-terminus become protonated and are easily ionized when analyzed by mass spectrometry. However, separating trypsin from the peptide fragments requires extra purification steps and can complicate the analysis if residual enzyme is left in the sample.
Immobilized trypsin provides a fast and convenient alternative for digesting proteins and separating the trypsin away from the peptide fragments. Promega has immobilized trypsin on cellulose particles and provided spin columns for easy protein digestion without trypsin contamination. An aliquot of particles is packed in the spin column and after three brief washing/equilibration steps, 20-500µg of protein is added to the column. A variety of protein samples can be digested without the need to concentrate or optimize digestion conditions. After a 30-minute digestion, peptides are easily separated from the immobilized trypsin by
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centrifugation as they flow through the spin column into the collection tube. Immobilized trypsin does not pass through the spin column frit, so only your peptides elute from the column. The purified peptides can be analyzed via mass spectrometry to identify the protein or assess any protein modification. Immobilized Trypsin enables: • ast Digestions: Complete F in 30 minutes. • calable Protocols: Adjusts S to protein concentration. • asy Setup: No shaking E or water baths required.
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Promega Australia - Monthly online Magazine - August 2010 edition Lees publicatie 2Home