罗氏蛋白酶抑制剂 EDTA-free说明书.pdf

1、www.roche-applied- 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 bact

2、eria Cat. No. 04 693 132 001 20 tablets y Version 04 Do not eat Content version: January 2013 Store at +2 to +8C 1. What this Product Does 1.1 Properties cOmplete, EDTA-free tablets inhibit a broad spectrum of serine and cysteine proteases. In contrast to other cOmplete tablets they do not contain E

3、DTA 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 sh

4、ow 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 prot

5、eases are not inhibited. 1.2 Contents 20 individually packed cOmplete Protease Inhibitor Cocktail Tablets in foil blisters. Each tablet is sufficient for a volume of 50 ml solution. 1.3 Stability The tablets are stable at +2 to +8C, stored dry, until expiration date. The stock solution is stable for

6、 12 weeks, stored at +2 to +8C, or at least for 12 weeks at 15 to 25 C. 1.4 Application 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

7、 stability or subsequent assays or purification methods (e.g., Immobilized Metal Chelate Affinity Chro- matography IMAC). 2. How to Use this Product 2.1 Handling Carefully push the tablet through the foil packaging using the base of your thumb (and not yout finger nail) to prevent the breakage of ta

8、b- lets. Do not eat the tablets. 2.2 Preparation 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 t

9、ablets 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 Info

10、rmation“). 2.3 Stock solution (25 conc.) Dissolve one cOmplete, EDTA-free tablet in 2 ml redist. H 2 O 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 effe

11、cted. 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 no

12、t 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 p

13、H 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

14、 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 rang

15、e, 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 immedia

16、te addition to the protease % inhibition after 60 min incubation (protease + cOmplete) at +15 to +25 C Pancreas- extract 0.02 7 .8 84% 94% Thermolysin (Metalloprote- ase) 0.0005 7 .8 7% 10% Chymotrypsin 0.002 0.02 7. 8 7. 8 90% 60% 100% 98% Trypsin 0.02 0.002 7. 8 7. 8 70% 81% 74% 86% Papain 0.33 7

17、.8 74% 87% 0113.04728661001Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany Contact and Support To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at: www.roche-applied- To call, write, fax, or email us

18、, visit the Roche Applied Science homepage, www.roche-applied-, and select your home country. Country- specific contact information will be displayed. Use the Product Search func- tion to find Instructions for Use and Material Safety Data Sheets. 4. Additional Information on this Product 4.1 Absorpt

19、ion A solution of one cOmplete, EDTA-free tablet dissolved in 50 ml H 2 O has an absorption of 0.08 at 280 nm. 4.2 Quality Control The inhibitory activity of each lot is tested with a concentrated pan- creas extract. Thereby, the proteolytic activitiy is typically inhibited by 94% after one hour (de

20、tection with universal protease substrate, casein, resorufin-labeled*). 4.3 Reference 1 North, M.J. (1969) in: Proteolytic Enzymes - A Practical Approach (Beynon, P.J. & Bond, J.S. eds.), IRL press Oxford, pp. 117-119. 4.4 Changes to previous version Ordering Information updated Editorial changes 5.

21、 Ordering Information For addional information, please visit www.roche-applied- cOmplete Protease Inhibitor Cocktail Tablets in EASYpacks: cOmplete Protease Inhibitor Cocktail Tablets in glass vials: cOmplete ULTRA Protease Inhibitor Cocktail Tablets: Product Pack size Cat. No. cOmplete 20 tablets i

22、n foil blisters (for 50 ml each) 04 693 116 001 cOmplete, Mini 30 tablets in foil blisters (for 10 ml each) 04 693 124 001 cOmplete, EDTA-free 20 tablets in foil blisters (for 50 ml each) 04 693 132 001 cOmplete, Mini, EDTA- free 30 tablets in foil blisters (for 10 ml each) 04 693 159 001 Product Pa

23、ck size Cat. No. cOmplete 20 tablets in a glass vial (for 50 ml each) 3 20 tablets in a glass vial (for 50 ml each) 11 697 498 001 11 836 145 001 cOmplete, Mini 25 tablets in a glass vial (for 10 ml each) 11 836 153 001 cOmplete, EDTA-free 20 tablets in a glass vial (for 50 ml each) 3 20 tablets in

24、a glass vial (for 50 ml each) 11 873 580 001 05 056 489 001 cOmplete, Mini, EDTA- free 25 tablets in a glass vial (for 10 ml each) 11 836 170 001 Product Pack size Cat. No. cOmplete ULTRA, Mini, EDTA-free, EASYpack 30 tablets in foil blisters (for 10 ml each) 05 892 791 001 cOmplete ULTRA, EDTA- fre

25、e, glass vials 2 10 tablets in glass vials (for 50 ml each) 6 10 tablets in glass vials (for 50 ml each) 05 892 953 001 05 892 953 001 cOmplete ULTRA, Mini, with EDTA, EASYpack 30 tablets in foil blisters (for 10 ml each) 05 892 970 001 cOmplete ULTRA, with EDTA, glass vials 2 10 tablets in glass vi

26、als (for 50 ml each) 6 10 tablets in glass vials (for 50 ml each) 05 892 988 001 06 538 304 001 PhosSTOP Phosphatase Inhibitor Cocktail Tablets: cOmplete His-Tag Purification: This combination comprises only the most important products related to the product described. Please refer to our latest cat

27、alog for our current product range or contact your local Roche Applied Science representative directly. Trademarks COMPLETE ia a trademark of Roche. All other product names and trademarks are the property of their respective owners. Regulatory Disclaimer For life science research only. Not for use i

28、n diagnostic procedures. Product Pack size Cat. No. PhosSTOP 10 tablets in foil blisters (for 10 ml each) 04 906 845 001 PhosSTOP 20 tablets in foil blisters (for 10 ml each) 04 906 837 001 Product Pack size Cat. No. cOmplete His-Tag Purification Resin 25 ml settled resin volume 200 ml settled resin volume 05 893 682 001 05 893 801 001