Application of a PEG/Salt Aqueous Two-Phase Partition System for The
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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. -
Ultracentrifugation Techniques for the Ordering of Nanoparticles
nanomaterials Review Ultracentrifugation Techniques for the Ordering of Nanoparticles Xufeng Xu 1,† and Helmut Cölfen 2,* 1 Laboratory of Physical Chemistry, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5612AE Eindhoven, The Netherlands; [email protected] 2 Physical Chemistry, University of Konstanz, Universitätsstraße 10, Box 714, 78457 Konstanz, Germany * Correspondence: [email protected] † Present address: Institute of Materials, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. Abstract: A centrifugal field can provide an external force for the ordering of nanoparticles. Especially with the knowledge from in-situ characterization by analytical (ultra)centrifugation, nanoparticle ordering can be rationally realized in preparative (ultra)centrifugation. This review summarizes the work back to the 1990s, where intuitive use of centrifugation was achieved for the fabrication of colloidal crystals to the very recent work where analytical (ultra)centrifugation is employed to tailor-make concentration gradients for advanced materials. This review is divided into three main parts. In the introduction part, the history of ordering microbeads in gravity is discussed and with the size of particles reduced to nanometers, a centrifugal field is necessary. In the next part, the research on the ordering of nanoparticles in analytical and preparative centrifugation in recent decades is described. In the last part, the applications of the functional materials, fabricated from centrifugation-induced nanoparticle superstructures are briefly discussed. Keywords: centrifugation; sedimentation; nanoparticle; concentration gradient; non-equilibrium process; superstructure; functional material Citation: Xu, X.; Cölfen, H. Ultracentrifugation Techniques for 1. Introduction the Ordering of Nanoparticles. 1.1. The Era of Microbeads in Gravity Nanomaterials 2021, 11, 333. -
Continuous-Flow Centrifugation to Collect Suspended Sediment for Chemical Analysis
1 Table 6. Compounds detected in both equipment blank samples and not in corresponding source blank samples or at concentrations greater than two times the corresponding source blank sample concentration. Prepared in cooperation with the National Water Quality Monitoring Council and [Source data: Appendix A, table A1; Conn and Black (2014, table A4); and Conn and others (2015, table A11). CAS Registry Number: Chemical Abstracts Service Washington State Department of(CAS) Ecology Registry Number® (RN) is a registered trademark of the American Chemical Society. CAS recommends the verifi cation of CASRNs through CAS Client ServicesSM. Method: EPA, U.S. Environmental Protection Agency’s SW 846; SIM, select ion monitoring. Unit: µg/kg, microgram per kilogram; ng/kg, nanogram per kilogram. Sample type: River samples were from the Puyallup River, Washington. Q, qualifi er (blank cells indicate an unqualifi ed detection). J, estimated, result between the Continuous-Flow Centrifugationdetection level and reporting level; toNJ, result Collect did not meet all quantitation Suspended criteria (an estimated maxiumum possible concentration is reported in Result column) U, not detected above the reporting level (reported in the Result column); UJ, not detected above the detection level (reported in the Result column). Abbreviations: na, not Sediment for Chemicalapplicable; Analysis PCBs, polychlorinated biphenyls] Sample type Chapter 6 of CAS River River Commercial Commercial Section D, Water Quality Parameter name Registry Method Unit source equipment -
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. -
Nanosep® Centrifugal Devices - Protocols for Use Nanosep® Centrifugal Devices - Protocols for Use
Contact Us: www.pall.com/contact Nanosep® Centrifugal Devices - Protocols for Use Nanosep® Centrifugal Devices - Protocols for Use Ultrafiltration Fundamentals Purification and Handling of DNA Fragments PCR: Before and After Protein Purification and Handling Miscellaneous Protocols Appendices References Ordering Information Ultrafiltration Fundamentals Background Ultrafiltration (UF) is a membrane separation technique used to separate extremely small particles and dissolved molecules in fluids. The primary basis for separation is molecular size, although other factors such as molecule shape and charge can also play a role. Molecules larger than the membrane pores will be retained at the surface of the membrane (not in the polymer matrix as they are retained in microporous membranes) and concentrated during the ultrafiltration process. Compared to non-membrane processes (chromatography, dialysis, solvent extraction, or centrifugation), ultrafiltration: Is far gentler to the molecules being processed. Does not require an organic extraction which may denature labile proteins. Maintains the ionic and pH milieu. Is fast and relatively inexpensive. Can be performed at low temperatures (for example, in the cold room). Is very efficient and can simultaneously concentrate and purify molecules. The retention properties of ultrafiltration membranes are expressed as Molecular Weight Cutoff (MWCO). This value refers to the approximate molecular weight (MW) of a dilute globular solute (i.e., a typical protein) which is 90% retained by the membrane. However, a molecule’s shape can have a direct effect on its retention by a membrane. For example, linear molecules like DNA may find their way through pores that will retain a globular species of the same molecular weight. There are three generic applications for ultrafiltration: 1. -
Centrifugal Hyperfiltration
TO: Technology Innovation Program Mail Stop 4750 National Institute of Standards and Technology 100 Bureau Drive Gaithersburg, MD 20899-4750 FM: Dirk Forman 125 S. St. Louis Lafayette Louisiana 70506 [email protected] Centrifugal Hyperfiltration A means to economically recover resources from saltwater and wastewater streams with lower the energy cost of producing freshwater and potable water from marginalized water resources. Introduction Reverse Osmosis (RO) is a filtration process for the removal of ionic and organic pollutants from wastewater. Today’s technology of utilization of this filtration process is by large array of high-pressure piping and pressure pumps. This process yields low volumes of filtrated output (permeate), utilizes large areas for pipe array and components and the concentration polarization and membrane fouling hinders the wide application of RO filtration process. Utilizing centrifugal forces and cross flow membranes has potential to change the standard (RO) process to one of portable and high volume water purification. AN AREA OF CRITICAL NATIONAL NEED Ensuring Future Water Supply: As the Nation’s population and economy grow, greater demands are being placed on freshwater resources. At the same time, temporary or permanent drought conditions and water access rights affect regional freshwater availability. Water needs threaten to outstrip available freshwater, now and in the future. Emerging contaminants that must either be removed from distributed water or converted to harmless forms of waste is also pressuring water quality, both in terms of decontamination and disinfection of water supplies. Food contaminations are often traced back to water contaminations, either in the field or in processing. Municipal waste streams and irrigation runoff waste resources that are not recovered.1 Over 97 percent of the Earth's water -- seawater and brackish groundwater -- is too salty to use for drinking water or agriculture. -
404 Sedimentation Handout
Sedimentation for Analysis and Separation of Macromolecules A ‘centrifugal force’ as experienced by particles in a centrifuge actually reflects the natural tendency of the particle to move in a straight line in the absence of external forces. If we view the experiment in the (fixed) reference frame of the laboratory, we observe that the particle is deflected from a linear trajectory by a centripetal force (directed toward the central axis of the rotor). However, when we consider the motion of the particle with respect to the (rotating) reference frame of the rotor, it appears that at any instant the particle experiences an outward or centrifugal force: Instantaneous Velocity (vtang) Centrifugal Acceleration Centripetal Acceleration As seen in laboratory As seen in (rotating) reference frame reference frame of rotor Ignoring buoyancy effects for the moment, the magnitude of the centrifugal force (which, in the rotating reference frame of the rotor, is as real as any other force) is m ⋅ v2 F = objecttan g =⋅mrω 2 centrif r object [1] where r is the distance from the axis of rotation, vtang is the linear velocity at any instant (in the direction perpendicular to the radial axis) and ω is the angular velocity (in radians sec-1 – note that 1 rpm = 2π///60 rad sec-1). The magnitude of this force can be compared to that of the gravitational force at the earth’s surface: Gm⋅()⋅() m = earth object Fgrav 2 re [2] -8 -1 3 -2 where G is the universal gravitational constant (= 6.67 x 10 g cm sec ) and re and me are the earth’s radius (= 6.37 x 108 cm) and mass (5.976 x 1027 g), respectively. -
Guide to Gel Filtration Or Size Exclusion Chromatography
Guide to Gel Filtration or Size Exclusion Chromatography www.harvardapparatus.com Table of Contents Introduction ........................................................................ 2-4 Size Fractionation .................................................................... 5 Buffer Sample Selection ........................................................ 6-7 Selection of Media and Size .................................................... 8 Gel Filtration SpinColumns .................................................... 9 Spehadex P-25 Applications .................................................. 10 Desalting Columns Applications ............................................ 11 P-2, P-6 and P-30 SpinColumns .......................................... 12 Ordering Information ............................................................ 13 Contact Information .............................................................. 14 Guide to Gel Filtration or Size Exclusion Chromatography 1 Introduction Gel Filtration Gel Filtration also called size-exclusion chromatography can be used for protein DNA purification, buffer exchange, desalting, or for group separation in which the sample is separated in two major groups. Gel Filtration is an easy to use method for separation of molecules with different molecular sizes, using mild conditions. Gel Filtration uses the size of molecules in solution to determine separation. SpinColumns have short media packing so the samples are separated by size, the large molecules travel out of the column with the void -
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. -
Recent Developments in Centrifuge Technology Harald Anlauf ∗ Universit¨Atkarlsruhe (TH), Institut F¨Urmvm, D-76128 Karlsruhe, Germany
First published in: Available online at www.sciencedirect.com Separation and Purification Technology 58 (2007) 242–246 Recent developments in centrifuge technology Harald Anlauf ∗ Universit¨atKarlsruhe (TH), Institut f¨urMVM, D-76128 Karlsruhe, Germany Abstract Centrifugation represents one of the main groups of mechanical particle-liquid separation processes. There are available various centrifuges for sedimentation and filtration, which are operating continuously or discontinuously. The tasks for centrifugal separation are very wide spread from liquid clarification and purification, particle thickening, solids demoistening, particle fractionating and sorting, solids washing, liquid–liquid separation to extraction of solids or liquids. Applications of centrifuges can be found in all kinds of industry, in environmental protection, water treatment, etc. In the centrifugal field mass forces are present during the separation process. This leads to specific advantages and disadvantages in comparison to competing separation processes. Research and development for centrifugal processes today has not coming to an end but several new ideas to use centrifugal forces for separation and new technical developments in this field can be observed. After description of some fundamentals and presentation of an overview of the technical variations some aspects of advantages and disadvantages of centrifuges in comparison to other separation techniques are discussed and examples for recent research and technical development as well as actual trends in the field of centrifugation are given. © 2007 Published by Elsevier B.V. Keywords: Solid–liquid separation; Centrifugation; Filtration; Sedimentation 1. Introduction like demonstrated in Fig. 3. Centrifuges are especially well suited to separate molecularly inmiscible liquids, whereby Centrifuges are representing one of the main techniques to frequently in addition solid particles are present. -
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. -
Manufacturing of Agarose-Based Chromatographic Media with Controlled Pore and Particle Size
MANUFACTURING OF AGAROSE-BASED CHROMATOGRAPHIC MEDIA WITH CONTROLLED PORE AND PARTICLE SIZE by Nicolas Ioannidis A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Chemical Engineering College of Engineering and Physical Sciences The University of Birmingham 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Chromatography remains the most commonly employed method for achieving high resolution separation of large-sized biomolecules, such as plasmid DNA, typically around 150-250 nm in diameter. Currently, fractionation of such entities is performed using stationary phases designed for protein purification, typically employing pore sizes of about 40 nm. This results into a severe underexploitation of the porous structure of the adsorbent as adsorption of plasmid DNA occurs almost exclusively on the outer surface of the adsorbent. In this study, the effect of two processing parameters, the ionic strength of agarose solution and quenching temperature, on the structure of the resulting particles was investigated. Three characterization methods, Atomic Force and cryo-Scanning Electron microscopy, as well as mechanical testing of single particles where used to quantify the effect of these parameters on the pore size/size distribution and mechanical properties of the adsorbent.