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Analysis Note

Protein determined by E1%/280

Application

Trypsin can be used to release adherent cells from tissue culture plates for passaging. Trypsin has been used in a study to assess the effects of macromolecular crowding on the structural stability of human α-lactalbumin. Trypsin has also been used in a study to investigate BN-PAGE analysis of Trichoderma harzianum secretome.

For trypsin digestion of peptides, use a ratio of about 1:100 to 1:20 for trypsin:peptide. The typical use for this product is in removing adherent cells from a culture surface. The concentration of trypsin necessary to dislodge cells from their substrate is dependent primarily on the cell type and the age of the culture. Trypsins have also been used for the re-suspension of cells during cell culture, in proteomics research for digestion of proteins and in various in-gel digestions. Additional applications include assessing crystallization by membrane-based techniques and in a study to determine that protein folding rates and yields can be limited by the presence of kinetic traps.

Biochem/physiol Actions

Trypsin cleaves peptides on the C-terminal side of lysine and arginine residues. The rate of hydrolysis of this reaction is slowed if an acidic residue is on either side of the cleavage site and hydrolysis is stopped if a proline residue is on the carboxyl side of the cleavage site. The optimal pH for trypsin activity is 7-9. Trypsin can also act to cleave ester and amide linkages of synthetic derivatives of amino acids. EDTA is added to trypsin solutions as a chelating agent that neutralizes calcium and magnesium ions that obscure the peptide bonds on which trypsin acts. Removing these ions increases the enzymatic activity. Serine protease inhibitors, including DFP, TLCK, APMSF, AEBSEF, and aprotinin, amongst others, will inhibit Trypsin.

General description

The trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the 8-anilinonaphthalene-1-sulfonate binding.

Other Notes

View more information on trypsin at www.sigma-aldrich.com/enzymeexplorer

Preparation Note

TPCK treated

Unit Definition

One BAEE unit will produce a A₂₅₃ of 0.001 per minute at pH 7.6 at 25°C using BAEE as a substrate.

One BAEE unit will produce a δA₂₅₃ of 0.001 per min at pH 7.6 at 25 °C using BAEE as substrate. Reaction volume = 3.2 ml (1 cm light path).