The Complete Guide for Protease Inhibition, Biol UMCS, VII semestr, Analiza biochemiczna
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//-->Roche Applied ScienceThe Complete Guide forProtease InhibitioncPmplete protection...cPmplete convenienceProteases are ubiquitous in all living cells. As soon as cells are disrupted, proteases arereleased and can quickly degrade any protein. This can drastically reduce the yield ofprotein during isolation and purification. Contaminating proteases can be inhibited byprotease inhibitors, thereby protecting the protein of interest from degradation.The Complete Guide for Protease Inhibition from Roche Applied Science is a comprehensiveresource to help you select the appropriate protease inhibitors for your applications. Thisbrochure includes information regarding the specificity, stability, effectiveness, and safety ofour protease inhibitors._____________________________Introduction to Protease Inhibitors _______________________________2-3Classes of Protease Inhibitors______________________________________3_____________________________cPmplete Protease Inhibitor Cocktail Tablets ______________________4-6_____________________________Pefabloc SC ______________________________________________________8Pefabloc SC PLUS ________________________________________________9_____________________________Protease Inhibitor Product Overviews___________________________10-14Protease Inhibitors Set ___________________________________________14Universal Protease Substrate _____________________________________14_____________________________Related Products and Ordering Information ________________________15Visitwww.roche-applied-science.com/proteaseinhibitorto learn more aboutprotease inhibition, and access technical information about our protease inhibitors,including tips on when and how to use the products.2|Protease Inhibitors from Roche Applied Science —cPmplete protection... cPmplete convenienceCells contain many different types of proteases. Therefore, mixtures of different inhibitors are needed for completeprotection of proteins during isolation and purification for subsequent experiments (e.g., western blotting, reporter geneanalysis, or protein interaction or activity assays).Roche Applied Science has extensive experience with the isolation, purification, and analysis of many different proteins,and with the best ways to protect these proteins from degradation. Choose an individual protease inhibitor for your specialapplication, or achieve broad-spectrum protection with the convenient cPmplete Protease Inhibitor Cocktail Tablets.Convenient —Choose from a wide range of water-soluble protease inhibitors for exceptional ease of use.Safe —Use non-toxic inhibitors, such as the cPmplete Protease Inhibitor Cocktail Tablets, Pefabloc SC, and PefablocSC PLUS, to achieve protease inhibition without risk to you, or those around you.Reliable —Count on our expertise in the production of high-quality, function-tested protease inhibitors that havebeen successfully used by researchers worldwide and referenced in thousands of publications.Classes of Protease Inhibitors available from Roche Applied ScienceGeneral inhibitors forProteaseInhibitorAprotininPefabloc SC and Pefabloc SC PLUSLeupeptin(inhibits serine and cysteine proteases with trypsin-like specificity)PMSFcPmplete, EDTA-freeProtease Inhibitor Cocktail Tablets*cPmpleteProtease Inhibitor Cocktail Tablets*2-Macroglobulin* When extractions or single-step isolations are necessary in theacidic pH range, include Pepstatin along with cPmplete tabletsto ensure aspartic (acid) protease inhibition.a) Contain serine and histidine in the active centerb) Contain cysteine (thiol, SH-) in the active centerc) Contain metal ions (e.g., Zn2+, Ca2+, Mn2+) in the active centerd) Contain aspartic (acidic) group in the active centerSerineproteasesaCysteineproteasesbE-64Metallo-proteasescPhosphoramidonBestatin(aminopeptidases)AsparticproteasesdPepstatinProtease-specificinhibitorsAntipain dihydrochlorideCalpain Inhibitor ICalpain Inhibitor IIChymostatinTLCKfor theinhibition of:Papain, Trypsin (Plasmin)Calpain I > Calpain IICalpain II > Calpain IChymotrypsinTrypsin, other serine and cysteine proteases(e.g., Bromelain, Ficin, Papain)TrypsinTrypsin-Inhibitor(chicken egg white, soybean)|3Simplify protease inhibition with convenience and reliabilityin a cPmplete tabletWhen isolating or purifying proteins, benefit fromthe ultimate in convenience – use cPmplete ProteaseInhibitor Cocktail Tablets and eliminate the time-consuming search for the right protease inhibitor.cPmplete is a proprietary blend of proteaseinhibitors, formulated as a ready-to-use water-soluble tablet. Simply add the convenient tablet toyour homogenization buffer, and instantly protectyour proteins against a broad range of proteases.Obtain the protection you need, with convenienceand reliability — choose our easy-to-use cPmpleteProtease Inhibitor Cocktail Tablets.Convenience■cPmpleteProteaseInhibitorCocktailTabletsConsistently inhibit a multitude of protease classes (Table 1), includingserine proteases, cysteine proteases, and metalloproteases.Inhibit proteolytic activity in extracts from almost any tissue or cell type,including animals, plants, yeast, bacteria, and fungi (for examples, see page 5).Choose from two available tablet sizes, with or without EDTA, for 10 or50 ml of lysate.Drop a tablet into your lysis buffer and eliminate the cumbersome job ofweighing small amounts of different protease inhibitors on an analyticalscale, and dissolving the mix in DMSO.■■■Reliability■■■Deliver consistent doses of protease inhibition.Obtain stable, non-toxic protection in aqueous buffers.Maintain the stability of metal-dependent proteins, and function of purification techniques(i.e., IMAC [immobilized metal affinity chromatography] for isolation of Poly-His-tagged proteins)by using EDTA-free cPmplete Protease Inhibitor Tablets.Count on cPmplete protection to eliminate the worryAchieve broad-spectrum protection with a singletablet. cPmplete Protease Inhibitor CocktailTablets eliminate the questions... and the doubt.Table 1: Inhibition of different proteases bycPmplete Protease Inhibitor Tablets.One cPmpletetablet was added per 50 ml incubation solution. Proteolyticactivity was determined with the Roche Applied ScienceUniversal Protease Substrate (casein, resorufin-labeled),Cat. No. 11 080 733 001. When extractions or single-stepisolations are necessary in the acid pH range, simplyinclude Pepstatin along with cPmplete tablets to ensureaspartic (acid) protease inhibition. All experiments wereperformed at room temperature.Source and concentrationof proteaseType ofprotease% Inhibitionimmediatelyafter addingcPmplete% Inhibition60 minutesafter addingcPmpleteChymotrypsin,1.5 µg/mlThermolysin,0.8 µg/mlPapain,1 mg/mlPronase,1.5 µg/mlPancreatic extract,1.5 µg/mlTrypsin,0.002 µg/mlSerineMetalloCysteineMixtureMixtureSerine97%99%95%88%87%93%97%100%73%99%99%73%4|Choose cPmplete inhibitionSelect the appropriate cPmplete Protease Inhibitor Cocktail Tablet for protection against unwanted protease activityin your application. Benefit from multiple tablet format options to meet your needs — choose from two tablet sizes(regular or mini), with or without EDTA. The choice is yours!Table 2: Choose the correctcPmplete tablet for yourapplication.ApplicationcPmpletetabletscPmplete,EDTA-freecPmplete,MinicPmplete,EDTA-freeInhibition during initialextraction steps(volumes > 50 ml)Inhibition during subsequentpurification protocols(volumes < 50 ml)Inhibition during subsequentpurification steps require freedivalent cations for furtherprocessing2Samples containing highmetalloproteolytic activity+++++++1+1++++++++++++++ Product of choice+ Can also be used0 Not recommended1 Preparation of stock solutions is recommended.2 Important, for example, with metal-chelatechromatography purification of Poly-His-taggedproteins, or protein samples used for signaltransduction research.cPmpleteProteaseInhibitorCocktailTabletsAchieve cPmplete successChoose our versatile cPmplete Protease Inhibitor Cocktail Tablets to provide the protease inhibition you need.Try the cPmplete Protease Inhibitor Cocktail Tablets today, and see how simple success can be. Your laboratory justisn’t complete without it.Some examples of cells, tissues, and organisms in which protease activity has been successfully inhibited withcPmplete tablets — as reported in scientific literature:■■Acintobacillus actinomycetemcomitansAdipocytes (mouse, rat)■Adrenal gland (PC-12, rat)■Bladder carcinoma cells (T24, human)■Bone marrow cells (mouse, human)■Bone osteosarcoma (U-2 OS, SaOs-2, human)■Brain neuroblastoma cells (SK-N-BE(2), human)■Brain tissue (bovine, mouse, rat, human)■Breast cancer cells (BT20, MCF7, human)■Bronchial Alveolar Lavage Fluid (mouse, rat)■Bronchial Biopsies (human)■Bronchial epithelial cell line (BZR, human)■Cardiomyocytes (mouse, rat)■Cervix adenocarcinoma (HeLa, human)■Cochlea (rat)■Colon carcinoma cells (T84, human)■Colorectal adenocarcinoma cells (CaCo-2) (human)■Colorectal and duodenal adenomas■Colorectal carcinoma cells (HCT-116, human)■Cortex (rat)■Dictyostelium (amoeba)■E. coli■Endothelial cell line■Epidermis (human)■Epithelial cell lines (human, bovine)■Fat (mouse)■Fibroblasts (human; NIH-3T3, MDTF, mouse)■Fibrosarcoma cell line (HT1080, human)■Fruit (tomato)■Glioblastoma cell line (U87MG)■Head (Drosophila)■■Heart (human, mouse, chicken)Hematopoietic cell lines (mouse, human)■Immature seed (soy)■Insect cell lines (Sf2, Sf21, Sf9, Tn5)■Keratinocytes (human)■Kidney (dog, human, mouse, rat, monkey,Xenopus)■Leaf (Arabidopsis)■Liver carcinoma cells (HepG2, Hep3B, human)■Liver tissue (mouse, rat,Xenopus)■Lung carcinoma cells (A549, human)■Lung homogenates (mouse,Xenopus)■Lung lavage fluid (mouse)■Luteal tissue (bovine)■Lymph nodes (mouse)■Lymphoblastoids (human)■Lymphocytes (Jurkat, human; WEHI 3b D, mouse;monkey)■Mammary carcinoma cells (MDA468, human)■Mammary epithelial cells (HMEC)■Mammary gland (mouse)■Mast cell line (human)■Monocyte cells (THP-1, human)■Muscle (Drosophila, human, mouse, rat, rabbit,Xenopus)■Neisseria gonorrhoeae■Neurons (rat)■Ovarian cancer (OVCAR-3, human)■Ovary cells (CHO, hamster)■Pancreas (mouse)■Parathyroid tissue (bovine)■Peripheral blood cells (BA/F3, mouse; CEM,HL-60, human)■Pichia pastoris■Placental labyrinth (mouse, rat)■Platelets (human)■Primary chondrocytes (human)■Primary lung cancer cells■Primary mast cells (mouse)■Primary neuronal cultures (mouse)■Prostate adenocarcinoma cells (PC-3, human)■Prostate carcinoma cells (DU-145 and LNCaP, human)■Pseudomonas■Rectal tissue (rabbit)■Renal cell carcinomas (human)■Reticulocyte lysate (rabbit)■Retina (mammalian)■Saccharomyces cerevisiae■Salivary gland (mouse)■Salmonella typhimurium■Seed (Arabidopsis)■Skin (human)■Spermatogenic cells (mouse)■Spinal cord (rat)■Spleen (mouse, rat,Xenopus)■Staphylococcus aureus■Streptococcus pneumoniae■Superior cervical ganglion (mouse)■Toxoplasma gondii■Umbilical vein endothelial cells (HUVEC, human)■Whole plant tissue|5
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