Proteolytic Enzymes Proteolytic Stock Storage Concentration Reaction Enzyme Solution Temperature in Reaction Buffer Temperature Pretreatment 1 2 Pronase 20mg/mL in H2O –20°C 1mg/mL 0.01M Tris 37°C Self-digestion (pH 7.8) 0.01M EDTA 0.5% SDS 3 Proteinase K 20mg/mL in H2O –20°C 50µg/mL 0.01M Tris 37° to 56°C None required (pH 7.8) 0.005M EDTA 0.5% SDS 1 Pronase is a mixture of serine and acid proteases isolated from Streptomyces griseus. 2 Self-digestion eliminates contamination with DNase and RNase. Self-digested Pronase is prepared by dissolving powdered Pronase in 10mM Tris•Cl (pH 7.5), 10mM NaCl to a final concentration of 20mg/mL and incubating for 1 hour at 37°C. Store the self-digested Pronase in small aliquots at –20°C in tightly capped tubes. 3 Proteinase K is a highly active protease of the subtilisin type that is purified from the mould Tritirachium album Limber. The enzyme has two binding sites for Ca2+, which lie some distance from the active site and are not directly involved in the catalytic mechanism. However, when Ca2+ is removed from the enzyme, approxi- mately 80% of the catalytic activity is lost because of long-range structural changes (Bajorath et al. Nature 1989; 337:481-484). Because the residual activity is usually sufficient to degrade proteins that commonly contaminate preparations of nucleic acids, digestion with Proteinase K is usually carried out in the presence of EDTA (to inhibit the action of Mg2+-dependent nucleases). However, to digest highly resistant proteins such as keratin, it may be necessary to use a buffer con- taining 1mM Ca2+ and no EDTA. At the end of the digestion, the Ca2+ should be chelated by addition of EGTA (pH 8.0) to a final concentration of 2mM before the nucleic acids are purified. From Molecular Cloning: A Laboratory Manual, second edition (1989). Fisher Scientific UK, Bishop Meadow Road, Loughborough, Leicestershire LE11 5RG Tel: +44 01509 231166 Fax: +44 01509 231893 E-mail: [email protected] www.fisher.co.uk.