Print For life science research only. Not for use in diagnostic procedures. Protease Inhibitors Set Cat. No. 11 206 893 001 1 set y Version 17 Content version: August 2014 Product description The set contains ten protease inhibitors. When used in varying combinations, a broad spectrum of proteases can be inhibited. For isolation and purification of proteins (e.g. enzymes), specific mixtures of inhibitors can be used, for example a cocktail of Aprotinin, E-64 or Leupeptin, EDTA, Pefabloc® SC and Pepstatin. Alternatively, our range of Complete Protease Inhibitor Cocktail Tablets provide a convenient and reliable solution for the inhibition of a broad spectrum of serine, cysteine, metalloproteases as well as calpains. Due to the optimized composition of the tablets they show excellent inhibition, effects and are therefore very well suited for the protection of proteins isolated from animal tissue, plants, yeast and bacteria. See below for ordering information. Table 1: Composition of the set: Suggested starting concentration ➀ Specificity of Inhibitors Solubility/ Stability Antipain-dihydrochloride (1) 3 mg 677.6 papain, trypsin soluble in H2O (20 mg/ml), methanol or DMSO ➁ 50 g/ml (74 M) Bestatin (1) 0.5 mg 308.4 amino peptidase, including aminopeptidase B., leucine aminopeptidase, tripeptide aminopeptidase soluble in 1 M HCI (20 mg/ml). Methanol (5 mg/ml) or 0.15 M NaCI (1 mg/ml). ➂ 40 g/ml (130 M) Chymostatin (3) 1 mg E-64 (4) 3 mg 607.7 -, -, -, -chymotrypsin 357.4 cysteine proteases soluble in glacial acetic acid (20 mg/ml) or DMSO ➁ soluble in 1:1 H2O/ethanol [v/v] (20 mg/ml) ➁ Leupeptin (3) 0.5 mg 475.6 serine and cysteine proteases such as plasmin, trypsin, papain, cathepsin B soluble in H2O (1 mg/ml) ➁ Pepstatin (3) 0.5 mg 685 9 aspartate proteases like pepsin, renin, cathepsin D, chymosin soluble in methanol (1.0 mg/ml) ➁ Phosphoramidon (3) 3 mg 543.5 soluble in metallo endopeptidases, specifically thermolysine, collagenase, metallo endo- H2O (20 mg/ml) ➁ proteinases. Pefabloc® SC (6) 20 mg 239.5 serine proteases, e.g. trypsin, chymotrypsin, plasmin, thrombin EDTA-Na2 (5) 10 mg 372.24 metalloproteases Inhibitor/Quantity Aprotinin (2) 0.5 mg ➀ Mr 6512 serine proteases 6– 60 g/ml (10–100 M) 0.5– 10 g/ml (1.4–28 M) 0.5– 5 g/ml (1– 10 M) 0.7 g/ml (1 M) 4– 330 g/ml (0.007– 0.6 mM) soluble in H2O (100 mg/ml) ➁ 0.1– 1 mg/ml (0.4– 4 mM) soluble in H2O to 0.5 M at pH 8-9.➁ 0.2-0.5 mg/ml (0.5-1.3 mM) soluble in H2O (10 mg/ml) ➂ (0.06– 2 g/ml (0.009- 0.3 M We recommend that these values be used only as a starting point. Final suitable concentrations must be determined empirically for each new system. ➁ Stock solutions are stable for 1 month stored at ⫺15 to ⫺25° C; storage in aliquots is recommended to avoid repeated thawing. ➂ Stock solutions are stable for 6 months stored at ⫺15 to ⫺25° C: storage in aliquots is recommended to avoid repeated thawing Note: The effectiveness of protease inhibition can be verified fast and sensitively with the general substrate for proteases casein, resorufin labeled* (7) For the different sources of biological material specifically adapted protease inhibitor cocktails have been found valuable for protein isolation (table 2): Table 2: Suggestions for inhibitor cocktalls for protein isolation modified from (8) and (9)]. Tissue/Organism Animal tissues Plant tissues Protozoa Slime moulds Yeast and fungi Bacteria Inhibitor Pefabloc SC EDTA Leupeptin Pepstatin Aprotinin Pefabloc SC Chymostatin EDTA Pefabloc SC Leupeptin Pefabloc SC Leupeptin TLCK Pefabloc SC Phenanthroline Leupeptin (9) Pepstatin Pefabloc SC EDTA Final concentration 1 mg/ml 0.5 mg/ml 10 g/ml 10 g/ml 1 g/ml 1 mg/ml 20 g/ml 0.5 mg/ml 1 mg/ml 10 g/ml 1 mg/ml 10 g/ml 0.04 mg/ml 1 mg/ml 1 mg/ml 3 g/ml 15 g/ml 1 mg/ml 0.5 mg/ml Stock medium water/buffer water/buffer water/buffer methanol water/buffer water/buffer DMSO water/buffer water/buffer water/buffer water/buffer water/buffer 1 mMol HCI water/buffer ethanol DMSA methanol water/buffer water/buffer Stock concentration 40 mg/ml 40 mg/ml 1 mg/ml 1 mg/ml 0.1 mg/ml 40 mg/ml 1 mg/ml 40 mg/ml 40 mg/ml 1 mg/ml 40 mg/ml 1 mg/ml 20 mg/ml 40 mg/ml 100 mg/ml 1 mg/ml 1 mg/ml 40 mg/ml 40 mg/ml If cysteine proteases are thought to cause problems in animal, plant or protozoa tissues, E-64 should be checked as additive (final concentration: 10 g/ml; stock solution: 1 mg/ml in water/buffer). 0814.11216 0150017 www.lifescience.roche.com Table 3: Protease inhibitors offered by Roche Diagnostics: Inhibitors Pack Size Cat. No. 20 tablets cpmplete Protease Inhibitor Cocktail Tablets (1 tablet for 50 ml) 3 × 20 tablets 11 697 498 001 11 836 145 001 cpmplete Mini (1 tablet for 10 ml) 25 tablets 11 836 153 001 cpmplete EDTA-free (1 tablet for 50 ml) 20 tablets 11 873 580 001 cpmplete Mini, EDTA-free (1 tablet for 10 ml) 25 tablets 11 836 170 001 (4-Amidinophenyl)-methanesulfonyl fluoride (APMSF) 10 mg 10 917 575 001 Antipain-dihydrochloride 10 mg 11 004 646 001 10 mg 50 mg 100 mg 10 236 624 001 10 981 532 001 11 583 794 001 Bestatin 10 mg 50 mg 10 874 515 001 11 359 070 001 Calpain inhibitor I 25 mg 11 086 090 001 Calpain inhibitor II 25 mg 11 086 103 001 L-1-Chloro-3-(4-tosylamido)-7amino-2-heptanone-hydrochloride (TLCK) 100 mg 250 mg 10 874 485 001 10 874 493 001 L-1-Chloro-3-(4-tosylamido)-4phenyl-2-butanone (TPCK) 1g 10 874 507 001 Chymostatin 10 mg 11 004 638 001 3,4-Dichloroisocoumarin 10 mg 10 973 840 001 E-64 5 mg 10 mg 25 mg 11 585 673 001 10 874 523 001 11 585 681 001 2000 ATU 11 110 276 001 5 mg 25 mg 50 mg 100 mg 11 017 101 001 11 017 128 001 11 034 626 001 11 529 048 001 Aprotinin from bovine lung Hirudin Leupeptin 2-Macroglobulin from bovine plasma Pefabloc® SC (AEBSF) Pefabloc® Plus Pepstatin from Streptomyces species Phenylmethylsulfonyl fluoride (PMSF) PMSF Plus Phosphoramidon Trypsin inhibitor from chicken egg white Trypsin inhibitor from soybean 25 inhibitor units References 1 2 3 4 5 6 7 8 9 Umezawa, H. (1982) Annu. Rev. Microbiol. 36, 75. Kassell, B. (1970) Methods Enzymol. 19, 844. Umezawa, H. (1976) Methods Enzymol. 45, 678. Hanada, K. et al. (1978) Agr. Biol. Chem. 42, 523. Maniatis, T. et al. (1982) Molecular Cloning: A Laboratory Manual, 446, Cold Spring Harbor Laboratory, New York. Markwardt, F. et al. (1973) Thromb. Res. 2, 343. Schickaneder, E. et al. (1988) Fresenius Z. Anal. Chem. 330, 360. North, M. J. (1989) in: Proteolytic enzymes — A practical approach p.117, Beynon, R. J. & Bond, J. S. (eds.), IRL-Press, Oxford. Jazwinski, S. M. (1990) Methods Enzymol. 182, 154. * available from Roche Diagnostics Changes to previous version Updated information regarding antipain-dihydrochloride specificity and suggested concentration. Trademarks COMPLETE is a trademark of ROCHE. PEFABLOC is a trademarks of Pentypharm AG, Basel, Switzerland. All other product names and trademarks are the property of their respective owners. Regulatory Disclaimer For life science research only. Not for use in diagnostic procedures. 10 602 442 001 100 mg 500 mg 1g 11 429 868 001 11 585 916 001 11 429 876 001 100 mg + additive 1 g + additive 11 873 601 001 2 mg 10 mg 50 mg 10 253 286 001 11 359 053 001 11 524 488 001 1g 10 g 25 g 10 236 608 001 10 837 091 001 11 359 061 001 1 g + additive 11 873 636 001 11 873 628 001 5 mg 10 874 531 001 1g 10 109 878 001 50 mg 500 mg 10 109 886 001 10 109 894 001 Roche Diagnostics offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our home page www.lifescience.roche.com. Contact and Support To ask questions, solve problems, suggest enhancements and report new applications, please visit our Online Technical Support Site. To call, write, fax, or email us, visit www.lifescience.roche.com, and select your home country. Country-specific contact information will be displayed. Roche Diagnostics GmbH Sandhofer Strasse 116 68305 Mannheim Germany