DOCSLIB.ORG

Protocols and Tips in Protein Purification

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Protocols and Tips in Protein Purification Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11. Protocol for accurate determination of concentration of pure protein 47 12. Calibration plot for gel filtration column 48 13. Ni-chelating cartridges : cleaning and recharging 48 14. SDS PAGE Sveta’s easy protocol 49 15. DTNB reaction 50 16. Refolding protocol 50 3 V. Charts and Tables 51 Ammonium sulphate –Sugar refractometer 53 NaCl-Sugar refractometer 54 Calibration plot for Hi-load Superdex 200 column 55 Calibration plot at various salt concentrations for Superdex200 GL column 56 Ammonium sulphate table 57 Properties of amino acids 58 VI. Appendix 1 59 1.Determination of protein concentration 59 2.How to concentrate proteins 64 3.How to store proteins 67 4.How to estimate purity of a protein preparation 68 VII. Appendix 2 69 1. Prepare your solutions right! 69 2. Gel filtration 70 Separation by molecule’s size 70 Calibration plot 71 Main reasons for “abnormal” mobility of some proteins during 72 gel filtration 3. Time saving tips 73 4. Golden rules in protein purification 74 VIII. Show cases 75 1. Two step purification protocol and a lesson to learn (BPSL 1549 (BLF1)) 75 2. It is easy with the coloured proteins! 79 Single-domain haemoglobin cgb from Campylobacter jejuni 79 Flavohemoprotein HMP from Escherichia coli 81 3. Almost typical three step purification (Glutamate Dehydrogenase from 85 Clostridium symbiosum) 4. Monitoring purification by enzyme activity (Glutamate Dehydrogenase 88 from Clostridium symbiosum) 5. Power and failure of dye pseudo affinity chromatography 91 Phenylalanine Dehydrogenase from Nocardia sp. 91 Glutamate Dehydrogenase from Clostridium symbiosum 97 6. DNA binding proteins 102 Typical Heparin protocol: RuvC from phage bIL67 102 Playing with solubility: LrpC from Bacillus subtilis 104 Heparin and solubility: FrmR from Escherichia coli 105 Real power of Heparin: Human FEN 108 In need of nuclease treatment: SfsA 111 7. Tagged proteins 116 Proper use of a His-tag: Human protein PARP1 expressed in E.coli 116 Removable tag: SAG19 118 Cleaver use of tag: TssA 121 8. Special cases 124 Phosphoglycerate Kinase (PGK) from Escherichia coli 124 Low affinity chromatography: BPSL1958 127 BacM from Myxococcus Xanthus 130 134 Perfect purity: IGPD from Arabidopsis thaliana 4 Abbreviations AA acrylamide APMSF 4-Amidinobenzylsulfonyl fluoride hydrochloride AS ammonium sulphate CBV column bed volume CE crude extract CFE cell free extract CM carboxymethyl DEAE diethylaminoethyl DTNB Dithionitrobenzole DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FF fast flow HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HIC hydrophobic interaction chromatography IEC ion exchange chromatography MES 2-morpholinoethanesulfonic acid MW molecular weight MWCO molecular weight cut off PBS phosphate saline buffer PS protamine sulphate PSA ammonium persulphate PAGE polyacrylamide gel electrophoresis Q quaternary ammonium S methyl sulphonate SDS sodium dodecyl sulphate SEC size exclusion chromatography or gel filtration SP sulphopropyl TEMED N,N,N',N' tetramethylethylenediamine TP target protein UV ultraviolet Contact details Dr. Svetlana Sedelnikova Department of Molecular Biology and Biotechnology The University of Sheffield Sheffield S10 2TN Please, contact me by e-mail [email protected] All your comments and questions are very welcome 5 With greatest respect and gratitude to my teachers Prof. Alexander Sergeevich Spirin and Prof. Marina Borisovna Garber and people of Institute of Protein Research, Pushchino and also to my employers Prof. David W. Rice and Department of Molecular Biology and Biotechnology of the University of Sheffield 6 I. Introduction Why do we need to purify protein in one day? After 25 years and 360 proteins it has become clear to me that for some proteins even one extra day under conditions normally used for protein purification can detrimentally affect their activity and crystallisation ability. Whilst the majority of proteins can survive long purification and can be kept and stored at 4ºC for days and even weeks with little loss in activity, it does them no harm to be purified in one day either. To achieve the goal of complete protein purification in one day you should move fast and choose appropriate protocols, avoiding long procedures such as dialysis, long centrifugations and slow chromatography. Below are some protocols and tips which help me to achieve this goal. My approach is based on classical combination of ion-exchange, hydrophobic and size-exclusion chromatography for natively (no tags) over-expressed proteins. Nowadays the high throughput approach dictates increasing use of tags in protein purification and sometimes classical methods are considered to be defunct. This is not the case. The most justified use of tags is for difficult expressions, such as expression of eukaryotic proteins in E.coli, expression of the domains of the proteins, toxic proteins, in the cases when tags help polypeptide chains to fold, in the cases when correct fold occurs only occasionally and so expression yield is hardly detectable. In such the cases tagged proteins often cannot be purified to the desirable purity by using just one tag affinity chromatography, so more purification steps are required anyway. We also have to remember that tag as well as being often useful in some cases it may be an obstacle that prevents correct proteins folding. I consider the recent trend to do all expressions in a tagged form is misleading . For most of enzymes and similar cytoplasmic proteins the chance to be successfully expressed is greater in the absence of the tag. My recent experience also revealed that if the fusion protein or the tag has to be removed from the target protein you may face a great problem either with protease underperforming or unspecific proteolysis leading to damage of the target protein. In this second edition I have included my recent experience with purification of the tagged proteins. Please note that this brochure is not a complete guide to protein purification and you should still read serious books on the theory of chromatography to become familiar with the subject. 7 SDS-PAGE equipment 8 II. General sequence of protein purification procedures Preparation of equipment and reagents For protein purification you need the equipment and reagents listed below: Sonicator or French Press Centrifuge, medium speed (30-70Kg, e.g. J-20 or Avanti J-25) Appropriate centrifugation tubes Chromatographic system comprising of, as a minimum, pump and fraction collector (normally system also includes UV monitor and recorder) Gradient mixer Spare pump or second chromatographic system (optional) Chromatographic columns packed with different types of matrixes Spectrophotometer VIS (340-800nm) or better UV/VIS (190-800nm) Bio-Rad Protein Assay Reagent Plastic cuvettes (1.6 ml) Stock solutions of salts and buffer components Concentrators (VivaSpin 20, 6 and 0.5ml) Low speed refrigerated centrifuge Filtration Device (such as Filter Holder or Stericup Filter Unit) Apparatus and solutions for SDS-PAGE (ideally precast gels) Refractometer (a pocket one for sucrose is fine) Pippetors and tips Eppendorf tubes Tubes for fraction collector 9 Preparation and use of stock solutions It is very convenient to have stock solutions of the main salts and buffers used during purification. Correctly and carefully prepared stock solutions can noticeably improve accuracy and reproducibility of the purification procedures. Stock solution preparation sequence When preparing stock solutions, follow the sequence below: Weigh powder → dissolve in about 80% of final required volume of ultra pure water→ adjust pH (for buffers and EDTA only) → adjust to final volume with ultra pure water → filter (also see poster in Apendix2) The most useful stock solutions in protein purification are: 5M NaCl 4M (NH4)2SO4 1M Tris-HCl, pH 8.0 o To prepare this solution you need to adjust the pH to 8.0 with concentrated HCl, this titration generates heat. However Tris buffer is temperature sensitive: raising the temperature by 3°C decreases pH by 0.1. To make a reproducible stock solution it is best to adjust pH on a water-ice bath with a thermometer placed in the solution, you are aiming for pH 8.0 at 20oC. A significant volume of concentrated HCl is required to adjust pH, therefore Tris powder should be dissolved in 60-70% of the final volume, pH adjusted to 8.0, then water added to final volume. Other stock solutions: 0.2M - 0.25M EDTA.
Load more

Recommended publications
  • Green Fluorescent Protein (GFP) Purification Student Manual
    Green Fluorescent Protein (GFP) Purification Student Manual "Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for "ice-minus" which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer." Michael Crichton - Jurassic Park Contents Lesson 1 Genetic Transformation Review—Finding the Green Fluorescent Molecule Lesson 2 Inoculation—Growing a Cell Culture Lesson 3 Purification Phase 1—Bacterial Concentration and Lysis Lesson 4 Purification Phase 2—Removing Bacterial Debris Lesson 5 Purification Phase 3—Protein Chromatography 26 Lesson 1 Finding the Green Fluorescent Molecule Genetic Transformation Review In Bio-Rad Kit 1, you performed a genetic transformation of E. coli bacterial cells. The results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet light. This is not a normal phenotype (characteristic) for E.coli. You were then asked to fig- ure out a way to determine which molecule was becoming fluorescent under UV light. After determining that the pGLO plasmid DNA was not responsible for the fluorescence under the UV light, you concluded that it was not the plasmid DNA that was fluorescing in response to the ultraviolet light within the cells. This then led to the next hypothesis that if it is not the DNA fluorescing when exposed to the UV light, then it must be a protein that the new DNA pro- duces within the cells. 1. Proteins. a. What is a protein? b.
    [Show full text]
  • Western Blotting Guidebook
    Western Blotting Guidebook Substrate Substrate Secondary Secondary Antibody Antibody Primary Primary Antibody Antibody Protein A Protein B 1 About Azure Biosystems At Azure Biosystems, we develop easy-to-use, high-performance imaging systems and high-quality reagents for life science research. By bringing a fresh approach to instrument design, technology, and user interface, we move past incremental improvements and go straight to innovations that substantially advance what a scientist can do. And in focusing on getting the highest quality data from these instruments—low backgrounds, sensitive detection, robust quantitation—we’ve created a line of reagents that consistently delivers reproducible results and streamlines workflows. Providing scientists around the globe with high-caliber products for life science research, Azure Biosystems’ innovations open the door to boundless scientific insights. Learn more at azurebiosystems.com. cSeries Imagers Sapphire Ao Absorbance Reagents & Biomolecular Imager Microplate Reader Blotting Accessories Corporate Headquarters 6747 Sierra Court Phone: (925) 307-7127 Please send purchase orders to: Suite A-B (9am–4pm Pacific time) [email protected] Dublin, CA 94568 To dial from outside of the US: For product inquiries, please email USA +1 925 307 7127 [email protected] FAX: (925) 905-1816 www.azurebiosystems.com • [email protected] Copyright © 2018 Azure Biosystems. All rights reserved. The Azure Biosystems logo, Azure Biosystems™, cSeries™, Sapphire™ and Radiance™ are trademarks of Azure Biosystems, Inc. More information about Azure Biosystems intellectual property assets, including patents, trademarks and copyrights, is available at www.azurebiosystems.com or by contacting us by phone or email. All other trademarks are property of their respective owners.
    [Show full text]
  • Water Specialist for the Oil & Gas Sector Ovivowater.Com
    Water Specialist for the Oil & Gas Sector creating value in water through innovation, creativity and expertise ovivowater.com © 2013 GLV Inc. All rights reserved. Ovivo - Industrial Markets Ovivo: A market Leader As society and the global economy demand more and more from water, there is a growing requirement for ever more applications to manage clean water, to create specialist process waters, to treat wastewater, to extract energy from wastewater and to champion the reuse of water. Ovivo - creating value in water through innovation, creativity and Many of the best known, most respected and reliable expertise in clean water, process brand names in the water and wastewater industries are part of Ovivo’s heritage. The combined strengths water, wastewater treatment, waste- of our brands and talents, including Brackett Green, to-energy and water reuse markets Caird & Rayner Clark, Christ Water Technology and across 5 continents. Eimco Water Technologies create one of the most comprehensive bank of technologies in the sector, one of the most impressive list of references, and practical application knowledge. Ovivo aims to become the water partner of choice for clients in the public and private sectors and the leading source of water expertise for engineers and consultants across the globe. Get in touch with some of the best brains in the business. For further information, visit ovivowater.com Ovivo - bringing water to life Copyright © 2013 GLV Inc. All rights reserved. Water Specialist for the Oil & Gas Sector Our Role in your Industry Oil and gas are precious resources. As an Ovivo develops and implements innovative integral element of oil and gas production, water solutions to meet the challenges of treating water and management is especially important.
    [Show full text]
  • The Art of Protein Purification
    1 The Art of Protein Purification William Ward Rutgers University, New Brunswick NJ, USA 1. Introduction Describing, in words, the details of protein purification to a relative novice in the field is not unlike explaining on paper the steps required to turn a set of colored oils into a beautiful pastoral scene on sheet of stretched canvas. Playing the oboe in a sophisticated metropolitan orchestra or performing a solo aria in a Gilbert & Sullivan operetta are accepted artistic endeavors that command great mastery of technique. Each of these art forms requires years of experience and endless experimentation and refinement of technique. Protein purification is no different. It is an art form. Like all other art forms, perfecting the art of protein purification requires a long apprenticeship. But, like all other art forms, protein purification is aesthetically rewarding to the practitioner. Every day brings new challenges, new insights, new hurdles, and new successes. Art is a process, not a destination. Protein purification fits the same definition. Perfecting the skills of protein purification can take many years of hands-on experience as well as periodic upgrading of those skills. Perhaps the most important part of protein purification is the set of pre-column steps that precede column chromatography. Pre- column steps are not covered as much in the protein purification literature as column chromatography, HPLC, and electrophoresis. So, I have chosen to focus much of my attention on the earlier stages of protein purification. More than column chromatography, pre-column steps are highly diverse and highly creative. Here the artistic aspects of protein purification are most apparent.
    [Show full text]
  • Anatomy and Physiology of Peritoneal Dialysis
    Anatomy and Physiology of Peritoneal Dialysis Isaac Teitelbaum, MD Professor of Medicine Director, Acute & Home Dialysis Programs University of Colorado Hospital Denver, Colorado •1 Outline • Peritoneal cavity as a dialysis system • Models of peritoneal transport • Physiology of peritoneal transport Inverse relationship between solute transport and ultrafiltration • Kinetics of peritoneal transport • Synthesis & Application • Middle Molecules Anatomy of The Peritoneum • The lining of the abdominal cavity • Two layers: parietal - lines the anterior wall and undersurface of the diaphragm - 20% of total SA; blood supply from abdominal wall visceral - covers the abdominal organs - 80% of total SA; blood supply from mesenteric aa and portal vv Gokal R, Textbook of PD, pp. 61-70 •3 Anatomy of The Peritoneum • Size 1.5 – 2 m2; approximates BSA • Highly Vascular • Semi-permeable/bi-directional • “Lymphatic” drainage through diaphragmatic stomata • Continuous with Fallopian Tubes in females Gokal R, Textbook of PD, pp. 61-70 1. The two main properties of the peritoneal membrane are: a. Semi permeable – this allows substances of certain sizes to move from an area of greater concentration to less concentration. b. Bi Directional - substances move in either direction across the membrane. 2. So-called “lymphatic” drainage refers to bulk flow from the peritoneal cavity back to the circulation. This actually occurs across tissues as well as lymphatics. As this is convective flow, dissolved solutes move with the fluid. Thus, fluid reabsorption results in loss of solute clearance as well as loss of fluid removal. 3. It is important to be aware of the continuity of the peritoneal cavity with the Fallopian tubes as retrograde menstruation- which may occur in any woman but goes undetected- will cause bloody dialysate and create concern in the PD patient.
    [Show full text]
  • Laboratory Filtration Product Guide
    Laboratory filtration Product guide gelifesciences.com 1 Welcome to Whatman filtration by GE Healthcare Life Sciences Our reputation, based on a solid foundation of expertise, enables us to support how healthcare is researched and delivered. In laboratories across the globe, the Whatman™ name is synonymous with quality, reliability, and ease of use. Our instinct for simplification accelerates the rate of discovery, reduces costs and saves time. Our products have a reputation for working right the first time – every time, which is why they are specified for the most exacting applications across a wide range of industries for people around the globe. Basic analytical testing In the vast and disparate world of analytical chemistry, Whatman products are used for basic laboratory processes that range from simple clarification to solvent extraction. Products range from filter papers, thimbles and Benchkote™ benchtop protectors, to membrane filters and phase separator papers. Food and beverage Our filter papers are used to prepare food samples prior to a wide range of analyses. Our syringe filters prevent fatty or particulate laden samples from damaging valuable equipment. Our membranes are used to test for harmful bacteria. Pharmaceutical Whatman products enable pharmaceutical companies to increase productivity. Mini-UniPrep™ syringeless filters and vials reduce HPLC sample preparation time and consumables usage, and track-etched and Anopore™ membranes are also vital to extruding liposomes for encasing and targeting drugs. Environmental monitoring Whatman products are cited in EPA, ASTM and ISO protocols for environmental monitoring. Whether it is detecting suspended solids in water, measuring air for dangerous particulates, or supporting asbestos analysis to maintain healthy spaces there is a Whatman filter that is central to the test.
    [Show full text]
  • Cellulose Nanopapers As Tight Aqueous Ultra- Filtration Membranes
    Cellulose nanopapers as tight aqueous ultra- filtration membranes Andreas Mautner1*, Koon-Yang Lee2, Tekla Tammelin3, Aji P. Mathew4, Alisyn J. Nedoma5, Kang Li5 and Alexander Bismarck1,6,* 1 Polymer & Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London, South Kensington Campus, SW7 2AZ London, United Kingdom 2 Department of Chemical Engineering, University College London, Torrington Place, WC1E 7JE London, United Kingdom 3 . VTT Technical Research Centre of Finland, Biologinkuja 7, FL-02044 Espoo, Finland 4 Division of Materials Science, Department of Engineering Sciences and Mathematics, Luleå University of Technology, 97187 Luleå, Sweden 5 Department of Chemical Engineering, Imperial College London, South Kensington Campus, SW7 2AZ London United Kingdom 6 Institute for Materials Chemistry & Research, Polymer & Composite Engineering (PaCE) Group, Faculty of Chemistry, University of Vienna, Währingerstr. 42, A-1090 Vienna, Austria * Corresponding authors: e-mail: [email protected], [email protected] Abstract: Recently, we have demonstrated the use of wood-derived nanocellulose papers, herein termed nanopapers, for organic solvent nanofiltration applications. In this study, we extend the use of these nanopapers to tight ultrafiltration (UF) membranes. The feasibility of such nanopaper-based UF membranes intended for use in water purification is shown. Four types of nanocelluloses, namely bacterial cellulose, wood-derived nanocellulose, TEMPO-oxidized cellulose nanofibrils and cellulose nanocrystals, were used as raw materials for the production of these nanopaper-based membranes. The resulting nanopapers exhibit a transmembrane permeance in the range of commercially available tight UF membranes with molecular weight cut-offs ranging from 6 to 25 kDa, which depends on the type of nanocellulose used.
    [Show full text]
  • PROTEIN a CHROMATOGRAPHY – the PROCESS ECONOMICS DRIVER in Mab MANUFACTURING
    PROCESS Application Note PROTEIN A CHROMATOGRAPHY – THE PROCESS ECONOMICS DRIVER IN mAb MANUFACTURING THE OPTIMIZATION OF THE PROTEIN A CAPTURE STEP IN DOWNSTREAM PROCESSING PLATFORMS CAN CONSIDER- ABLY IMPROVE PROCESS EFFICIENCY AND ECONOMICS OF INDUSTRIAL ANTIBODY MANUFACTURING. PARAMETERS LIKE RESIN REUSE AND ITS CAPACITY CONTRIBUTE CONSIDERABLY TO THE PRODUCTION COSTS. THE USE OF A HIGH CAPACITY PROTEIN A RESIN CAN IMPROVE THE PROCESS EFFICIENCY AND ECONOMICS. THIS PAPER PRESENTS THE KEY FEATURES OF A NEW CAUSTIC STABLE PROTEIN A RESIN PROVIDING EXTREMELY HIGH IgG BINDING CAPACITIES. Biopharmaceuticals represent an ever growing important Protein A affinity resins are dominating the Cost of Goods part of the pharmaceutical industry. The market for recom- (COGs) of mAb manufacturing. Bioreactors at the 10.000 L binant proteins exceeded $ 100 billion in 2011 with a scale operating at a titer of about 1g/L typically generate contribution of 45% sales by monoclonal antibodies (mAbs) costs of $ 4-5 million (2). Therefore the Protein A capturing (1). The introduction of the first mAb biosimilars in Europe step is the key driver to improve process economics. Besides raised the competitive pressure in an increasingly crowded the capacity of the resin, life time and cycle numbers market place. The industry faces challenges, such as patent significantly contribute to the production costs in mAb expirations accompanied by approvals of corresponding manufacturing. biosimilars, failures in clinical trials/rejections or the refusal of health insurers to pay for new drugs. Today, the IgG binding capacities of most Protein A resins are in the range of 30-50 g/L, offering significant advantages These challenges force the industry to minimize risk and time- for the processing of high-titer feedstreams when compared to-market and to proceed more cautiously.
    [Show full text]
  • Liquid / Solids Separation in Wastewater Treatment & Biosolids Dewatering
    LIQUID / SOLIDS SEPARATION IN WASTEWATER TREATMENT & BIOSOLIDS DEWATERING Chemical Products Lab Testing Plant Trials LIQUID / SOLIDS SEPARATION APPLICATIONS Influent Water Clarification Process Water Recycling Primary Wastewater Clarification Secondary Clarification Sludge Thickening Sludge Dewatering LIQUID / SOLIDS SEPARATION UNIT OPERATIONS Clarifiers (Many Types) WATER Filters (Many Types) OR WASTE Dissolved Air Flotation Units WATER Induced Air/Gas Flotation Units Belt Presses Centrifuges SLUDGE Screw Presses DEWATERING Plate and Frame Presses Vacuum Filters (Rotary & Horizontal) LIQUID / SOLIDS SEPARATION PRODUCT TYPES Coagulants (+) Low Mol Wt Organic Inorganic Blended Flocculants (+ , ---, 0 ) High Mol Wt Dry Emulsion Solution OilOil----FreeFree Flocculants COAGULANTS AND FLOCCULANTS Act on Insoluble Particles in Water Oils, Grease, Blood, Insoluble Organics, Clay, Silicates, Metal Oxides/Hydroxides Dirt, Dust, Rust & Metal Filings Can Act on Charged Organic Compounds Anionic Surfactants, Soaps & Dispersants Do Not Act on Most Dissolved Solids Salts, Acids, Nonionic Surfactants, Ammonia or Soluble Organic Compounds such as Sugar, Alcohols, etc. SUSPENSION CHEMISTRY THE KEY TO EFFECTIVE LIQUID / SOLIDS SEPARATION SUSPENDED SOLIDS VARIABLES Surface Charge MOST Charge Density Particle Size IMPORTANCE Composition Particle Density Particle Shape LEAST MICROSCOPIC FORCES ELECTROSTATIC BROWNIAN VAN DER WAALS GRAVITY Colloidal Particle in Water +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Almost all Particles +++
    [Show full text]
  • Water Treatment for Hemodialysis
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Hong Kong JournalJ Nephrol of 2001;3(1):7-14.Nephrology MKH TONG, et al 2001;3(1):7-14. REVIEW ARTICLE Water treatment for hemodialysis Matthew Ka-Hang TONG1, Wei WANG2, Tze-Hoi KWAN1, Lawrence CHAN2, Tak-Cheung AU1 1Department of Medicine, Tuen Mun Hospital, Tuen Mun, Hong Kong; 2Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA. Abstract Water treatment plays a vital role in the delivery of safe and effective hemodialysis (HD). Ensuring that water quality meets the American Association for the Advancement of Medical Instrumentation standards and recommendations (or equivalent) is necessary to reduce the incidence of chemical hazards and endotoxemia associated with the use of water for HD. This review will discuss the principles of water treatment for HD, the essential components of water purification, the recommended system monitoring and maintenance procedures, and some of the historical incidents of adverse reactions that resulted from the use of contaminated dialysis water. Key words: Dialysis solutions, Hemodialysis (HD), Pyrogenic reactions, Reverse osmosis (RO), Water purification ! !"#$%&'()*EeaF !"#$%&'()*+,-./0123456789%: ea !"#$%&'()*+,-*./0123456789ea !"#$%& !"#$%&'()#*+,-./0123456789:;<=> INTRODUCTION can build up to toxic levels, causing long-term physical Since the early 1960s, hemodialysis (HD) has been harm, and other substances are immediately toxic and increasingly used for the treatment of acute renal failure can cause death. A normal person also encounter these and end-stage renal failure. Technologic advances in contaminants through drinking water, but the healthy dialyzer membranes, dialysis machines and vascular kidney is able to remove these substances.
    [Show full text]
  • Mixtures Are a Combination 3.1 of Two Or More Substances MIXTURES
    Mixtures are a combination 3.1 of two or more substances MIXTURES A solution is a solute dissolved 3.2 in a solvent Mixtures can be separated according 3.3 to their physical properties 3.4 Mixtures can be separated according 3 to their size and mass What if? The different boiling points of liquids Case mix 3.5 can be used to separate mixtures What you need: a variety of different pencil cases (size, shape, colour) What to do: 1 Place all the pencil cases in Solubility can be used to one pile. 3.6 separate mixtures 2 List your pencil case’s properties that will allow it to DRAFT be identified easily (e.g. colour, shape, size and weight). 3 Give the list to another student. Can they identify your case by Waste water is a mixture that using the list? 3.7 can be separated What if? » What if you were blindfolded? Could you still find your pencil case? » What if the pencil cases were too small to feel? How could Materials recovery facilities you identify yours? 3.8 separate mixtures » What if all the pencil cases were exactly the same? Would it still be a mixture? Mixtures are a 3.1 combination of two or more substances Consider the things around you. Perhaps they are made of wood, glass or plastic. Wood, glass and plastic are all mixtures – each of these materials is made up of two or more substances. Some materials are pure substances. A pure substance is one where all the particles are identical.
    [Show full text]
  • Purification of the Recombinant Green Fluorescent Protein from Tobacco
    Dong et al. BMC Biotechnology (2019) 19:86 https://doi.org/10.1186/s12896-019-0590-y RESEARCH ARTICLE Open Access Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography Jie Dong1,2,3, Xiangzhen Ding1,2,3 and Sheng Wang1,2,3* Abstract Background: The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. Results: The recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. Conclusions: The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP. Keywords: Green fluorescent protein, Plant virus, Transient gene expression, Aqueous two-phase system, Hydrophobic interaction chromatography Background Through the application of DNA recombinant technol- Green fluorescent protein (GFP) was originally derived ogy, GFP has successfully been produced by a variety of from jellyfish Aequorea victoria species, which exhibit an hosts [5].
    [Show full text]