ReAction No.3 Product Selection September 2015

Transcription

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. Description Target rotease Class / Target Enzymes Order No. Quantity AEBSF Hydrochloride BioChemica Antipain Dihydrochloride BioChemica Aprotinin BioChemica Bestatin Hydrochloride BioChemica E-64 N-(trans-Epoxysuccinyl)- L-leucine-4- guanidinobutylamide Leupeptin Hemisulfate epstatin A rotease Inhibitor Cocktail 6 His-Tag rot serine proteases, thrombin, chymotrypsin, kallikrein, plasmin, proteinase K, trypsin serine/cysteine proteases, trypsin, papain, cathepsin B serine proteases, trypsin, chymotrypsin, kallikrein, plasmin amino-peptidase B, leucine amino-peptidase, tripeptide amino-peptidase, aminopeptidases of the cell surface cysteine proteases, papain, bromelain, calpain, cathepsin B, H, L, tumor cathepsin, Streptococcus protease, ficin serine/cysteine proteases, plasmin, trypsin, papain, cathepsin B, thrombin, calpain acid proteases, aspartic proteases, pepsin, cathepsin D, renin, HIV- und MMTV-proteases 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 A1421,0250 A1421,0500 A2129,0010 A2129,0025 A2132,0025 A2132, mg 500 mg 10 mg 25 mg 25 mg 100 mg A2137, mg A2157,0005 A2157, mg 10 mg A2183, mg A2205, mg 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 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. A B Chemicals structures of (A) AEBSF hydrochloride and (B) Bestatin hydrochloride Quick check 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 Biochemistry 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 enicillin- Streptomycin, 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 CO 2 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 50 ml of Incuwater-Clean per 5 liters of water. Incuwater-Clean A5219, ml 100x concentrate solution Incubator-Clean A5230, ml

5 Biochemistry 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 plifect 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 A3672,0250 L-Glutamine for cell culture A3704,0100 A3704, ml 250 ml 100 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 Histofix Substitute of Formaldehyde ready to use for clinical diagnosis ml 5 L 1000 ml 5 L

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 com- plies 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. 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). Gravimetric method (ISO 1211) Ammonia 25 % (as NH3) (Reag. US, h. Eur.) for analysis Amyl Alcohol according to NF V for analysis ml, ml MILK Butyrometer method (Gerber method) (ISO 2446) Gravimetric method (ISO 1735) CHEESE Butyrometer method (Van Gulik method) (ISO 3433) 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 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 ml, ml, ml, ml, ml, ml, ml,

8 8 ReAction No.3 Reagents for food analysis Gravimetric method + 10 g sample (milk) or 1 3 g sample (cheese) + 2 ml NH 3 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! Interface! Solvent Close and shake! phase Aqueous Fat extraction flask (Mojonnier type) Distill off the solvent and weigh Butyrometer method Fat collection flask (i.e. boiling flask) + 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 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. 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. Sample catalyst rotein (-N) + H " SO (NH ) SO + CO + H O 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 (NH 3 ) by reacting with hydroxyl ions (OH - ) of excess sodium hydroxide: (NH 4 ) " SO + 2 NaOH 2NH 2 (gas) + Na SO + 2H O 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. 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. Digestion Distillation Titration Organic nitrogen is converted into NH 4 + NH 3 is distilled and retained in a receiver vessel 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 NH 4 + into NH 3 4. NH 3 is condensed 3. NH 3 is dragged by bubbling steam 5. NH 3 retained by an acid solution (boric acid, sulfuric acid or hydrochloric acid) 1. Sample already digested with sulfuric acid Kjeldahl equipment 6. Finally NH 3 is titrated with sulfuric acid or sodium hydroxide volumetric solution Digestion Kjeldahl Catalyst (Cu-Se) (1.5% CuSO4.5H2O + 2% Se) tablets, according to Wieninger Kjeldahl Catalyst (Cu) (6.25% in CuSO4.5H2O) tablets, according to Directive 93/28/EEC g (1000 tablets of 1.0 g) 3.5 kg (1000 tablets of 3.5 g) 5 kg (1000 tablets of 5.0 g) kg (1000 tablets of 4.0 g) Sulfuric Acid 98% ml, 2.5 L Silicone antifoaming liquid ml, 250 ml, 500 ml Distillation and retention of NH 3 Sodium Hydroxide solution 40% w/w ml, 5 L, 10 L, 25 L Sodium Hydroxide solution 32% w/v ml,, 10 L, 25 L Boric Acid solution 4% ml, 5 L, 25 L Ammonia Fixative solution 1% L, 25 L Sulfuric Acid 0.25 mol/l (0.5N) ml, 2.5 L, 10 L Titration Sodium Hydroxide 0.1 mol/l (0.1N) ml,, 10 L Sulfuric Acid 0.05 mol/l (0.1N) ml, 5 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 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 S. enteritidis ATCC Incubation at 35 ± 2ºC / 24 hours Serological and Biochemical confirmation Non-selective pre-enrichment Buffered eptone Water (ISO 6579:2002) (repared Bottles) x100 ml Buffered eptone Water (ISO 6579:2002) (repared Bottles) x 3 L Buffered eptone Water (ISO 6579:2002) (Dehydrated Culture Media) g Selective enrichment Rappaport-Vassiliadis (RVS) Broth (ISO 6579:2002) (Dehydrated Culture Media) g Tetrathionate Broth Base acc. to Muller-Kauffmann (Dehydrated Culture Media) g 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 Other Non-ISO Media Chromogenic Salmonella Agar Hektoen Enteric Agar etc S. enteritidisis ATCC13076 Incubation at 35ºC ± 2ºC 24 hours E.coli ATCC25922 Incubation at 35ºC ± 2ºC 24 hours Chromogenic Salmonella Agar (Dehydrated Culture Media) g Salmonella Chromogenic Media (repared late (Ø 90 mm)) plates

12 A83; hoto: istockphoto.com Henk Badenhorst For more information please visit