www.roche-applied-science.com For life science research only. Not for use in diagnostic procedures. cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets provided in EASYpack For the complete inhibition of serine and cysteine proteases during extractions from animal and plant tissues or cells, yeast and bacteria Cat. No. 04 693 132 001 20 tabletsy Version 04 Do not eat Content version: January 2013 Store at +2 to +8C 1.What this Product Does 1.1Properties cOmplete, EDTA-free tablets inhibit a broad spectrum of serine and cysteine proteases. In contrast to other cOmplete tablets they do not contain EDTA leaving the stability and the function of metal depen- dant proteins uneffected. The affinity purification of Poly-His tagged fusionproteins via IMAC (immobilized metal affinity chromatogra- phy) is also facilitated (no dialysis necessary). Due to the optimized composition of the tablets they show excellent inhibition of serine and cysteine proteases and are therefore very well suited for the protection of proteins isolated from animal tis- sues, plants, yeast and bacteria. cOmplete, EDTA-free tablets con- tain both irreversible and reversible protease inhibitors. Metallo and aspartic proteases are not inhibited. 1.2Contents 20 individually packed cOmplete Protease Inhibitor Cocktail Tablets in foil blisters. Each tablet is sufficient for a volume of 50 ml solution. 1.3Stability The tablets are stable at +2 to +8C, stored dry, until expiration date. The stock solution is stable for 12 weeks, stored at +2 to +8C, or at least for 12 weeks at ?15 to ?25 C. 1.4Application Used for the inhibition of serine and cysteine proteases in bacterial, yeast, plant and animal cell extracts. For the inhibition of proteolytic activity in larger volumes, in which EDTA may interfere with protein stability or subsequent assays or purification methods (e.g., Immobilized Metal Chelate Affinity Chro- matography IMAC). 2.How to Use this Product 2.1Handling Carefully push the tablet through the foil packaging using the base of your thumb (and not yout finger nail) to prevent the breakage of tab- lets. Do not eat the tablets. 2.2Preparation of Working Solutions One cOmplete, EDTA-free tablet is sufficient for the inhibition of the proteolytic activity in 50 ml extraction solution. If very high proteolytic activity is present, one tablet should be used for 25 ml extraction buf- fer. The tablets can be added directly to the extraction medium. Alter- natively a stock solution (25 conc.) can be prepared. If it is necessary to inhibit proteolytic activity in a smaller volume we recommend to use cOmplete, Mini, EDTA-free tablets (1 tablet for 10 ml extraction solution, see “Ordering Information“). 2.3Stock solution (25 conc.) Dissolve one cOmplete, EDTA-free tablet in 2 ml redist. H2O or in 100 mM phosphate buffer, pH 7.0. 3.Results cOmplete, EDTA-free tablets are applied to stabilize those extracts where the stability or activity of metal containing proteins should not be effected. Since EDTA interferes with IMAC (immobilized metal affinity chromatography) cOmplete, EDTA-free tablets are preferen- cially used in the isolation process of Poly-His tagged fusion proteins. cOmplete, EDTA-free tablets inhibit efficiently serine and cysteine pro- teases in a broad range but not metalloproteases. Occasionally, aspartic proteases (“acid proteases“) can interfere upon isolations from animal tissues or bacterial extracts (1). These prote- ases, however, exhibit pronounced activities only at low pH values. If extraction or single isolation steps have to be performed at this pH range the addition of pepstatin* is recommended to inhibit aspartic protease activity as a precaution. Typical values for the inhibition of different proteases and protease mixtures by cOmplete, EDTA-free tablets are shown in table 1. Table 1: Inhibition of different proteases by cOmplete Protease Inhi- bitor Tablets. One cOmplete, EDTA-free tablet was added per 50 ml incubation solution. Pro- teolytic activity was determined with the Roche Applied Science Universal Pro- tease Substrate (casein, resorufin-labeled*). When extractions or single-step isolations are necessary in the acid pH range, simply include pepstatin* along with cOmplete, EDTA-free tablets to ensure aspartic (acid) protease inhibition. All experiments were performed at +15 to +25C. * available from Roche Applied Science Protease resp. protease mixture Enzyme concen- tration (mg/ml) pH- value % inhibition after immediate addition to the protease % inhibition after 60 min incubation (protease + cOmplete) at +15 to +25 C Pancreas- extract 0.027.884%94% Thermolysin (Metalloprote- ase) 0.00057.87%10% Chymotrypsin0.002 0.02 7.8 7.8 90% 60% 100% 98% Trypsin0.02 0.002 7.8 7.8 70% 81% 74% 86% Papain0.337.874%87% 0113.04728661001 Print Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany Contact and Support To ask questions, solve problems, suggest enhancements or report new appli