ReAction No.3 Product Selection September 2015
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1 ReAction No.3 roduct Selection September 2015
2 2 ReAction No.3 Biochemistry rotease Inhibitors In all eukaryotic cells and bacteria a large number of proteases are located in various compartments, the cytosol, mitochondria, vacuoles, lysosomes, ER, or in the extracellular space. Intracellular proteases are essential regulators in the synthesis, activation and degradation of proteins. Extracellular or secreted proteases are most prominent in the intestinal tract of animals or as a part of the blood-clotting cascade. Accordingly, different tissues or organisms contain different sets of proteases. Knowledge of the protease set of a particular expression system enables researchers to combat protease activity throughout the procedure of purification and analysis of proteins. Target rotease Class / Target Enzymes AEBSF Hydrochloride BioChemica serine proteases, thrombin, chymotrypsin, kallikrein, plasmin, proteinase K, trypsin A1421,0250 A1421, mg 500 mg Antipain Dihydrochloride BioChemica serine/cysteine proteases, trypsin, papain, cathepsin B A2129,0010 A2129, mg 25 mg Aprotinin BioChemica serine proteases, trypsin, chymotrypsin, kallikrein, plasmin A2132,0025 A2132, mg 100 mg Bestatin Hydrochloride BioChemica amino-peptidase B, leucine amino-peptidase, tripeptide amino-peptidase, aminopeptidases of the cell surface A2137, mg E-64 N-(trans-Epoxysuccinyl)L-leucine-4guanidinobutylamide cysteine proteases, papain, bromelain, calpain, cathepsin B, H, L, tumor cathepsin, Streptococcus protease, ficin A2157,0005 A2157, mg 10 mg Leupeptin Hemisulfate serine/cysteine proteases, plasmin, trypsin, papain, cathepsin B, thrombin, calpain A2183, mg epstatin A acid proteases, aspartic proteases, pepsin, cathepsin D, renin, HIV- und MMTV-proteases A2205, mg rotease Inhibitor Cocktail 6 His-Tag rot mixture of inhibitors optimized for the purification of His-Tag proteins from cell extracts. Contains AEBSF, Bestatin, E-64, epstatin A, and hosphoramidon. Ready-to-use solution A7802, ml
3 Biochemistry ReAction No.3 3 How protease inhibitors work rotease inhibitors often resemble structural elements of the respective protease substrate. They bind more or less specifically either reversibly or irreversibly to their target protease. Two examples are A. AEBSF acts by irreversible inhibition by sulfonylation of a functional group A in the active center of the protease. B. Bestatin resembles a he-leu substrate dipeptide, but the first residue contains an α-hydroxy group resulting in competitive active site-directed inhibition. B Chemicals structures of (A) AEBSF hydrochloride and (B) Bestatin hydrochloride Rapid test for His-Tag protein expression Rapid detection of over-expressed His-Tag proteins using Gold AB-HisDetect Gold AB-HisDetect is a Gold conjugated anti-his-tag antibody solution. His-tagged proteins are specifically stained in pink directly on the blot. The stain gives a quantitative and permanent signal. Stained protein is seen with the naked eye and may be recorded simply with a camera or scanner. Detection limit within the pico-molar range comparable to ECL. Western blot staining within only 60 minutes Does not require luminescence detection Gold AB-HisDetect A9747, ml
4 4 ReAction No.3 Cell Culture Novel antibiotics for Cell Culture an alternative to en:strep CellCultureGuard is a combination of novel antibiotics that prevent microbial contaminations in animal and human cell cultures. roviding protection against extra- and intracellular growing bacteria (including mycoplasma), protozoa and fungi (yeast), CellCultureGuard replaces conventional antibiotics such as enicillinstreptomycin, Gentamycin or Amphotericin B. Application: CellCultureGuard is a 100-fold concentrated sterile solution. Add 1 ml of CellCultureGuard to 100 ml of cell culture medium. CellCultureGuard is stable in cell cultures for 7 days at 37 C. Store at -20 C. CellCultureGuard A8906, ml Incubator-Clean and Incuwater-Clean Incubator-Clean solution is a spray to prevent contamination of cell culture incubators. The solution prevents growth of fungi, molds, bacteria (and spores), mycoplasma and eliminates many viruses. Application: Spray CO2 incubators every other week using Incubator-Clean. The active ingredients are quaternary benzylammonium compounds. Incubator -Clean is non-toxic and biodegradable (it does not contain mercury, formaldehyde, phenol or alcohol). Incuwater-Clean. The water required to create the humidity is a source of contamination which disperses in the incubator. Application: Change the incubator water with fresh sterile water every two to four weeks, adding 10 ml of Incuwater-Clean per 1 liter of water. Incuwater-Clean 100x concentrate solution A5219, ml Incubator-Clean A5230, ml
5 Molecular and Cell Biology ReAction No.3 5 Get the most out of your transfections AppliFect LowTox is an improved transfection reagent for liposome-mediated transfection of mammalian cells. For highest transfection efficiency at minimal cytotoxic effects. AppliFect LowTox A9027, ml A B Medium + DNA (or RNA) Medium + AppliFect LowTox combine Add complex to cells Incubate cells Test gene activity after 1 3 days A. Outline of the fast Transfection protocol using AppliFect LowTox B. Overlay of transmitted light and fluorescence signal of HEK293T cells transfected with an egf fusion protein using AppliFect LowTox. Related products for cell culture AC-Trypsin Solution for cell culture A8336, ml Trypsin 1 : 250 from porcine pancreas A4148, g Dimethyl Sulfoxide for cell culture A3672,0100 A3672, ml 250 ml L-Glutamine for cell culture A3704,0100 A3704, g 500 g BS tablets ph 7.4 (for 100 ml) A9162, tabs BS tablets ph 7.4 (for 200 ml) A9177, tabs
6 6 ReAction No.3 Microscopy & Histology Reagents for Clinical Diagnosis Histofix Substitute of Formaldehyde Commission Regulation (EU) 605/2014 and amendment No. 2015/491 establish new rules for classification and labeling of dangerous substances and precautionary statements and use of these substances. Formaldehyde is, among others, one of the substances affected by this new European legislation, so that we recommend the use of alternative substances and decreasing the exposure times of the users to this product. anreac AppliChem has developed a new product that assists to achieve the objectives of the new regulations: The new Histofix Substitute of Formaldehyde is a fixing agent commonly used in histological techniques, based on glyoxal, which reduces the health hazards for users. Main advantages: It is not carcinogenic. Flexible. It adapts to different fixation procedures: product concentrated and ready to use Convenient and practical packaging (1 and 5 liters) depending on the needs of each user. Less pungent odour than formaldehyde. Histofix Substitute of Formaldehyde for clinical diagnosis ml 5L Histofix Substitute of Formaldehyde ready to use for clinical diagnosis ml 5L
7 Reagents for food analysis ReAction No.3 7 Fat content determination in Dairy products During their handling and processing, milk and dairy products are subjected to stringent analytical controls to guarantee their composition and quality. Commission Regulation (EC) No 273/2008, of 5 March 2008, lays down parameters to determine the reference limits and methods for the chemical, physical and microbiological analysis, and for the organoleptic evaluation of milk and dairy products. One of the most important parameters is fat content. Fat content determination is of great importance because: This parameter impacts on the price paid per liter of milk. It is used to determine if a sample of milk or cheese complies with established legal values. It is necessary to know its value to classify the milk for the preparation of derivatives. There are different methods for determining the content of fat in milk and cheese. hoto: istockphoto.com ValentynVolkov Two typical methods are presented: 1. Mojonnier method: gravimetric method that uses organic solvents to extract fat. Subsequently the solvent is evaporated and the fat is determined by weighing the dry fatty extract. 2. Gerber method: volumetric method that uses chemical reagents (sulfuric acid, detergents) to achieve the breaking of the emulsion and the fat separation. Then the fat content is measured in special flask (butyrometer). MILK Ammonia 25 % (as NH3) (Reag. US, h. Eur.) for analysis Amyl Alcohol according to NF V for analysis ml Diethyl Ether stabilized with ~6 ppm of BHT (Reag. h. Eur.) for analysis, ACS, ISO Ethanol 96 % v/v for analysis, ACS Hydrochloric Acid 25 % for analysis, ISO Isoamyl Alcohol according to Gerber for analysis L, 25 L etroleum Ether C for analysis, ACS, ISO Sulfuric Acid % according to Gerber for analysis Sulfuric Acid 62 % (d= 1.522) according to Van Gulik for analysis Gravimetric method (ISO 1211) CHEESE Butyrometer Gravimetric method method (Gerber (ISO 1735) method) (ISO 2446) Butyrometer method (Van Gulik method) (ISO 3433)
8 8 ReAction No.3 Reagents for food analysis Gravimetric method Interface Solvent! Close and shake Fat extraction flask (Mojonnier type)!! + 10 g sample (milk) or 1 3 g sample (cheese) + 2 ml NH3 25 % (for milk) or ml HCl 25 % (for cheese) and heat + 10 ml ethanol + 2 drops Congo Red + 25 ml Diethyl Ether + 25 ml etroleum Ether Aqueous phase Distill off the solvent and weigh Fat collection flask (i.e. boiling flask) Butyrometer method + 10 ml Sulfuric Acid (90-91 % (d=1.820 g/ml) for milk or 62 % (d=1.522 g/ml) for cheese) + 11 ml sample (milk) or 3 g sample (cheese) + 1 ml Isomyl Alcohol for milk or 1 ml Amyl Alcohol for cheese Close and Shake vigorously Centrifugate Leave in bath (65 66 ºC) approx. 3 min. } Direct read: Fat column from milk or cheese (4.5-1= 3.5 % of fat) Sulfuric acid Butyrometer DIN12836 for determining the fat content according to Gerber
9 Reagents for food analysis ReAction No.3 9 Kjeldahl Nitrogen determination roteins are of indispensable nutritional value for humans and animals and are contained in the most common foodstuffs (juices, dairy products, meat, cereals, feed). In fact, the protein content is one of the important parameters declared on nutrition fact labels. The ammonia distilled is dragged by bubbling steam, condensed and collected in a receiver containing an acid solution. This acid can be boric acid solution, sulfuric acid or hydrochloric acid that captures the ammonia forming solvated ammonium ions. The Kjeldahl method allows the calculation of protein contents in food samples. It is based on the nitrogen determination which is a general constituent of all proteins. 3. Titration: The concentration of the captured ammonium ions in the boric acid is determined by means of an acid base titration commonly using standard solutions of sodium hydroxide. The scope of Kjeldahl nitrogen determinations today also includes applications in the fields of environmental analysis, research and development, pharmaceutical, chemical and cosmetics industries. The Kjeldahl procedure involves three major steps: 1. Digestion: The sample is mixed with sulfuric acid at temperatures between 340 and 370 ºC. The organic bonded nitrogen is converted into ammonium ions. Organic carbon and hydrogen form carbon dioxide and water. otassium sulfate and catalysts are added in order to increase the boiling point of sulfuric acid and to decrease the digestion time. catalyst Sample rotein (-N) + H2SO4 (NH4)2SO4 + CO2 + H2O " 2. Distillation: rior to the distillation the acidic sample is neutralized by means of concentrated sodium hydroxide solution (NaOH). In the distillation step the ammonium ions are converted into ammonia gas (NH3) by reacting with hydroxyl ions (OH ) of excess sodium hydroxide: (NH4)2SO4 + 2 NaOH " 2NH3 (gas) + Na2SO4 + 2H2O Digestion Organic nitrogen is converted + into NH4 Distillation NH3 is distilled and retained in a receiver vessel hoto: istockphoto.com haoliang Titration Nitrogen is determined
10 10 ReAction No.3 Reagents for food analysis Reagents used in Kjeldahl analysis 2. Sodium hydroxide solution is added to + neutralize the ph and to convert NH4 into NH3 4. NH3 is condensed 5. NH3 retained by an acid solution (boric acid, sulfuric acid or hydrochloric acid) 3. NH3 is dragged by bubbling steam 1. Sample already digested with sulfuric acid Kjeldahl equipment 6. Finally NH3 is titrated with sulfuric acid or sodium hydroxide volumetric solution g (1000 tablets of 1.0 g) Digestion Kjeldahl Catalyst (Cu-Se) (1.5% CuSO4.5H2O + 2% Se) tablets, according to Wieninger 3.5 kg (1000 tablets of 3.5 g) 5 kg (1000 tablets of 5.0 g) Kjeldahl Catalyst (Cu) (6.25% in CuSO4.5H2O) tablets, according to Directive 93/28/EEC kg (1000 tablets of 4.0 g) Sulfuric Acid 98% L Silicone antifoaming liquid ml, 250 ml, 500 ml Sodium Hydroxide solution 40% w/w L, 10 L, 25 L Sodium Hydroxide solution 32% w/v , 10 L, 25 L Boric Acid solution 4% L, 25 L Ammonia Fixative solution 1% L, 25 L Sulfuric Acid 0.25 mol/l (0.5N) L, 10 L Sodium Hydroxide 0.1 mol/l (0.1N) , 10 L Sulfuric Acid 0.05 mol/l (0.1N) L, 10 L Indicator 4.8, Mixed (Methyl Red-Bromocresol Green) ml Indicator 4.4, Mixed (Methyl Red-Methylene Blue) ml Methyl Red solution 0.1% ml Distillation and retention of NH3 Titration Other reagents available!
11 Microbiology ReAction No.3 11 Determination of Salmonella sp. according to ISO 6579:2002 in food products XLD and Salmonella Chromogenic agar According to the ISO 6579:2002 standard, the procedure for Salmonella analysis consists in a non-selective preenrichment in Buffered eptone Water and then reseeding in two selective enrichment broths (Tetrationate according to Mueller-Kauffman and Rappaport-Vassiliadis). The next step consists in inoculating by streaking onto selective agars such as the XLD medium and others (Hektoen, Salmonella and Shigella, Chromogenic for Salmonella, etc.). The suspected colonies must be confirmed with biochemical and serological tests. rocedure According to the ISO 6579:2002 Standard Non-selective pre-enrichment Buffered eptone Water ISO Selective enrichment MKTTn Broth or Rappaport-Vassiliadis Broth ISO Differential isolation on Selective Agar XLD Agar ISO 4 S. enteritidis ATCC Incubation at 35 ± 2ºC / 24 hours Chromogenic Salmonella Agar Hektoen Enteric Agar etc S. enteritidis ATCC13076 Incubation at 35ºC ± 2ºC 24 hours E.coli ATCC25922 Incubation at 35ºC ± 2ºC 24 hours Serological and Biochemical confirmation x100 ml Non-selective pre-enrichment Buffered eptone Water (ISO 6579:2002) (repared Bottles) Buffered eptone Water (ISO 6579:2002) (repared Bottles) x 3L Buffered eptone Water (ISO 6579:2002) (Dehydrated Culture Media) g Rappaport-Vassiliadis (RVS) Broth (ISO 6579:2002) (Dehydrated Culture Media) g Tetrathionate Broth Base acc. to Muller-Kauffmann (Dehydrated Culture Media) g Selective enrichment Differential isolation on Selective Agar XLD Agar (ISO 6579:2002) (Dehydrated Culture Media) g XLD Agar (ISO 6579:2002) (repared late (Ø 90 mm)) plates Chromogenic Salmonella Agar (Dehydrated Culture Media) g Salmonella Chromogenic Media (repared late (Ø 90 mm)) plates Other Non-ISO Media
12 hoto: istockphoto.com Henk Badenhorst A83; For more information please visit
ReAction No.3 Product Selection September 2015
ReAction No.3 roduct Selection September 2015 2 ReAction No.3 Biochemistry rotease Inhibitors In all eukaryotic cells and bacteria a large number of proteases are located in various compartments, the cytosol,
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