Monoliths in Bioprocess Technology
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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. -
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. -
Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications Using Monolithic Column Technology
Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications using Monolithic Column Technology A thesis submitted to University College London for the degree of Doctor of Engineering by David Isaac William Vincent The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Gordon Street London WC1H 0AH 1 I, David Vincent confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed……………….………………………Date……………………………………… 2 Acknowledgements I would like to thank my supervisor Dr Yuhong Zhou for her invaluable feedback and support over the course of this EngD. Without her continuous encouragement this work would likely never have been completed. I would also like to thank my Advisor Prof. Ajoy Velayudhan for his help and guidance. Many a long discussion was had on the intricacies of vaccinia viral purification which were thought provoking, inspirational but also really good fun. I would also like to express my gratitude to the staff and students at UCL. There are too many to mention everyone, but in particular I would like to thank Mrs Ludmila Ruban, Miss Dhushy Stanislaus and Dr Tarit Mukhopadhyay for their support, especially in setting up the vaccine lab in the department which I could benefit from in my final year. I would also like to thank Dr Patricia Perez Esteban for her support, friendship and bioprocess wisdom. I would like to express my gratitude to BIA Separations and the EPSRC for sponsoring this work. Without this financial support this work would not have been possible. -
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. -
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. -
BIA Separations CIM Monolithic Columns for Purification and Analytics of Biomolecules
BIA Separations CIM Monolithic Columns for Purification and Analytics of Biomolecules Lidija Urbas, PhD [email protected] Outline • BIA Separations • Chromatography – Monolithic Chromatography – Design of monolithic columns • DSP applications • PAT columns and applications: – Case study: combining DSP and PAT of Adenoviruses • Conclusions Ajdovščina BIA Separations • BIA Separations was founded in September 1998. • Headquarters in Austria, R&D and Production in Slovenia. BIA Separations • BIA Separations was founded in September 1998. • Headquarters in Austria, R&D and Production in Slovenia. • BIA Separations USA established in September 2007 - sales and tech support office. • BIA Separations China established in January 2011 - sales and tech support office. • 90 employees world wide • Main focus: To develop and sell methacrylate monolithic columns & develop methods and processes for large biomolecules separation and purification. BIA Separations – Products and Services CIM Contract monolithic Research columns Laboratory Method development and Technical Support Important Milestones • 2004: First monolith used for the industrial cGMP purification for plasmid DNA at Boehringer Ingelheim provide 15-fold increase in productivity • 2006: First cGMP production of a vaccine (influenza) using CIM® • 2008: OEM Partnership with Agilent Technologies – develop and produce analytical monolithic columns for PAT • In 2011 BIA Separations was awarded by KAPPA-Health as a model SME in the EU Co-funded research projects • 2012: co-marketing and -
Post-Polymerization Modifications of Polymeric Monolithic Columns: a Review
Chromatography 2014, 1, 24-53; doi:10.3390/chromatography1010024 OPEN ACCESS chromatography ISSN 2227-9075 www.mdpi.com/journal/chromatography Review Post-Polymerization Modifications of Polymeric Monolithic Columns: A Review Sinéad Currivan and Pavel Jandera * Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 532 10, Czech Republic; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +420-460-037-023. Received: 18 December 2013; in revised form: 16 January 2014 / Accepted: 17 January 2014 / Published: 10 February 2014 Abstract: The vast cache of methods used in polymeric monolithic column modification is presented herein, with specific attention to post-polymerization modification reactions. The modification of polymeric monolithic columns is defined and can include the modification of pre-existing surface groups, the addition of polymeric chains or indeed the addition of structures such as nano-particles and nano-structures. The use of these modifications can result in the specific patterning of monoliths, useful in microfluidic device design or in the investigation of modification optimization. Keywords: polymer monoliths; modifications; HPLC; CEC; grafting; click chemistry; hypercross-linking; nano-particles List of Abbreviations: 2-acrylamido-2-methyl-1-propanesulphonic acid AMPS, α, α’-azoisobutyronitrile AIBN, adenosine triphosphate ATP, atom transfer radical polymerization ATRP, benzoyl peroxide -
Capillary Electrochromatography for Analysis of Proteins and Metalloproteinases
San Jose State University SJSU ScholarWorks Master's Theses Master's Theses and Graduate Research 2008 Capillary electrochromatography for analysis of proteins and metalloproteinases Vasudha Salgotra San Jose State University Follow this and additional works at: https://scholarworks.sjsu.edu/etd_theses Recommended Citation Salgotra, Vasudha, "Capillary electrochromatography for analysis of proteins and metalloproteinases" (2008). Master's Theses. 3572. DOI: https://doi.org/10.31979/etd.m48v-cr76 https://scholarworks.sjsu.edu/etd_theses/3572 This Thesis is brought to you for free and open access by the Master's Theses and Graduate Research at SJSU ScholarWorks. It has been accepted for inclusion in Master's Theses by an authorized administrator of SJSU ScholarWorks. For more information, please contact [email protected]. CAPILLARY ELECTROCHROMATOGRAPHY FOR ANALYSIS OF PROTEINS AND METALLOPROTEINASES A Thesis Presented to The Faculty of the Department of Chemistry San Jose State University In Partial fulfillment of the Requirements for the Degree Master of Science by Vasudha Salgotra August 2008 UMI Number: 1459696 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 1459696 Copyright 2008 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. -
High Performance Capillary Electrophoresis
High Performance Capillary Electrophoresis A Primer A Primer High Performance Capillary Electrophoresis Second completely revised edition by Henk H. Lauer and Gerard P. Rozing Foreword Electrophoresis was born at the beginning of nineteenth century, even earlier than chromatography. It has taken a long development path, which in the early days was framed by names like Kohlrausch, Tiselius and later Everaerts. At the beginning of eighties in the last century, Jorgenson cause a paradigm shift by executing electrophoresis in a very thin capillary with inner diameter less then 100 μm leading to capillary electrophoresis. Since then, capillary electrophoresis has been constantly improved and has become a routine technique. Instrument manufacturers like Agilent Technologies have developed instrumentation for capillary electrophoresis serving many analytical laboratories reliably for many years. After a decline at the beginning of this decade, the capillary electrophoresis market is recovering strongly. Especially, hyphenation with mass spectrometers has been very fruitful and brought a remarkable ability to unravel complex structures of biomolecules with invincible speed and sensitivity. And there are several modes of capillary electrophoresis, which make it useful for separation of really wide class of compounds, such as small ions, peptides, proteins, DNA fragments both sequencing and restriction ones, and even complete cells or uncharged molecules. When compared with liquid chromatography capillary electrophoresis has one big advantage: the separation process takes place in a rather simple environment – in homogeneous solutions or in a polymeric sieving network and in a separation space with simple geometry, like a cylindrical channel. The equations describing completely movement of matter in space and time can be than easily formulated and solved by advanced mathematical methods. -
Two-Dimensional Electrochromatography/Capillary Electrophoresis on a Microchip
Frederick Conference on Capillary Electrophoresis, Hood College, Frederick, Maryland October 16-28, 2000 Two-Dimensional Electrochromatography/Capillary Electrophoresis on a Microchip Norbert Gottschlich, Stephen C. Jacobson, Christopher T. Culbertson, and J. Michael Ramsey Oak Ridge National Laboratory, Oak Ridge, TN 37831-6142 Two-dimensional (2D) separation methods for the analysis of complex protein or peptide mixtures have mostly been performed on planar gels using isoelectric focusing and polyacrylamide gel electrophoresis (IEF-PAGE). However, these techniques can be slow and labor intensive. Recently, several column-based two-dimensional separation schemes have been developed to reduce the analysis time. Another approach is to use microfluidic devices (microchips) that enable very fast and efficient separations. Furthermore, microchips are relatively easy to operate and allow the manipulation of very small sample volumes with minimal dead volumes between interconnecting channels. These features are especially useful for the development of multidimensional separations. We will report a comprehensive two-dimensional separation system on a microfabricated device that utilizes open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension is operated under isocratic conditions, and its effluent is injected into the second dimension every few seconds. A 25 cm long separation channel with spiral geometry for the first dimension, chemically modified with octadecylsilane, is coupled to a 1.2 cm long straight separation channel for the second dimension. Fluorescently labeled products from tryptic digests of proteins are analyzed in 13 minutes with this system. Research sponsored by Office of Research and Development, U.S. Department of Energy, under contract DE-AC05-00OR22725 with Oak Ridge National Laboratory, managed and operated by UT-Battelle, LLC.. -
Analytical Separations Using Packed and Open-Tubular Capillary Electrochromatography" (2004)
Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2004 Analytical separations using packed and open- tubular capillary electrochromatography Constantina P. Kapnissi Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Chemistry Commons Recommended Citation Kapnissi, Constantina P., "Analytical separations using packed and open-tubular capillary electrochromatography" (2004). LSU Doctoral Dissertations. 595. https://digitalcommons.lsu.edu/gradschool_dissertations/595 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. ANALYTICAL SEPARATIONS USING PACKED AND OPEN- TUBULAR CAPILLARY ELECTROCHROMATOGRAPHY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry by Constantina P. Kapnissi B.S., University of Cyprus, 1999 August 2004 Copyright 2004 Constantina Panayioti Kapnissi All rights reserved ii DEDICATION I would like to dedicate this work to my husband Andreas Christodoulou, my parents Panayiotis and Eleni Kapnissi, and my sisters Erasmia, Panayiota and Stella Kapnissi. I want to thank all of you for helping me, in your own way, to finally make one of my dreams come true. Thank you for encouraging me to continue and achieve my goals. Thank you for your endless love, support, and motivation. Andreas, thank you for your continuous patience and for being there for me whenever I needed you during this difficult time. -
Development of Microfluidic Electrophoresis Separation Methods for Calmodulin Binding Proteins
Development of Microfluidic Electrophoresis Separation Methods for Calmodulin Binding Proteins By 2014 Thushara Nalin Samarasinghe Submitted to the graduate degree program in Chemistry and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________ Dr. Carey Johnson (Chairperson) ________________________________ Dr. Susan Lunte ________________________________ Dr. Michael Johnson ________________________________ Dr. David Weis ________________________________ Dr. Prajna Dhar Date Defended: February 27th , 2015 The Dissertation Committee for Thushara Nalin Samarasinghe certifies that this is the approved version of the following dissertation: Development of Microfluidic Electrophoresis Separation Methods for Calmodulin Binding Proteins ________________________________ Dr. Carey Johnson (Chairperson) Date approved: February 27th , 2015 II Abstract Calmodulin (CaM) is a Ca2+ signaling protein that regulates more than 100 different enzymes in many intracellular pathways. Investigation of this complex CaM-binding “interactome” requires a sensitive and rapid screening mechanism. The objective of this research work is to develop a highly sensitive fluorescence-based detection method coupled with microfluidic electrophoresis separation assay for CBPs. A functional microfluidic separation platform with red laser-induced fluorescent detection was developed. It is a semi-automated system with integrated functional modules, a separation module,