Electrophoresis SUBJECT FORENSIC SCIENCE
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Impact Factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 REVIEW ON: ELECTROPHORESIS: METHOD for PROTEIN SEPARATION Shindedipa
Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 Pharma Science Monitor 7(2),Apr-Jun 2016 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com REVIEW ON: ELECTROPHORESIS: METHOD FOR PROTEIN SEPARATION ShindeDipa V.*, JasminaSurati Department of Quality Assurance, Shree NaranjibhaiLalbhai Patel College of Pharmacy,Umrakh -394 345,Bardoli, Gujarat, India. ABSTRACT Electrophoresis is one of the widely used techniques in molecular biochemistry, microbiology, biomedical research. It is a type of protein separation method .It is one of the highly efficient techniques of analysis and sole method for separation of proteins for western blot, RNA studies etc. It is a both qualitative and quantitative analysis technique. Separation depend upon electrophoretic mobility.Electrophoresis technique are of various type like Moving boundary electrophoresis ,Zone electrophoresis ,Affinity electrophoresis ,Pulsed field electrophoresis ,Dielectrophoresis.this technique mainly used in antibiotic analysis,vaccine analysis DNA analysis and protein analysis as well as fingerprint analysis. KEYWORDS:Electrophoresis, Electrophoretic mobility,Zone Electrophoresis, Moving boundary Electrophoresis, Dielectricphoresis. INTRODUCTION Electrophoresis is a physical method of analysis based on the migration of electrically charged proteins, colloids, molecules or other particles dissolved or dispersed in an electrolyte solution in the direction of the electrode bearing the opposite polarity when an electric current is passed through it. Separations may be conducted in systems without support phases (such as free solution separation in capillary electrophoresis) or in stabilising media such as thin-later plates, filins or gels. The electrophoretic mobility is the rate of movement in metres per second of the charged particles under the action of an electric field of I volt per metre and is expressed in square metres per volt second. -
"O *A*\., with (Fig- Coomassiebrilliant Blue 1 \Ar*.H'cre Hols H Ure 1)
A Convenient Procedure For storage, PAGE gels are often ide (PVDF) membranesare commonly treatedin one of two ways. They can be usedmatrices for transferblotting (12). for Transfer Blotting of placedin sealedplastic bags containing Here we describe a convenient, in- CoomassieBIue Stained 7Voaeueous acetic acid (9). More com- expensive method for long-terrn non- monly, these gels are photographed photographic storage of the informa- Proteins from PAGE and dried. In lieu of storage,when fur- tion present in electrophoresisgels Gels to Transparencies ther manipulationof the proteinsin the stainedwith dyes basedon blotting the gels is to be done,the patternis trans- stainedgels onto commercial transpar- encies of the type used in preparing ABSTRACT ferred to blotting membranes. Transfer blotting of proteins from viewgraphs for overhead projection. Proteins stained with CoomassieBril- PAGE gels to other films hasbeen ex- The dye-stained transparenciesfaith- liant Blue 1 were transferred effectively by aminedpreviously (7). Electroblotting, fully replicate the information present blotting frctm polvctcryktmidegel electro- diffusion and convection blotting are in the originally stained gel. They are (PAGE) phoresis gels to transparenciesof three common techniquesfor transfer convenientto store.They are also ex- plain-paper the Npe used in copiers. The of proteins (7). Nitrocellulose mem- cellent substratesfor use with gel scan- detuils rl' the original electropherogram branes, diazotrzedpapers, nylon-based ners, since they are flat, dimensionally were retained on transfer and did not fade membranesand polyvinylidene fluor- stable,and haveno color themselves. over a period of three years. Both the pro- tein and the associateddye transfer; how- ever, protein does not transfer in the ab- .\ence of dye. -
Power and Limitations of Electrophoretic Separations in Proteomics Strategies
Power and limitations of electrophoretic separations in proteomics strategies Thierry. Rabilloud 1,2, Ali R.Vaezzadeh 3 , Noelle Potier 4, Cécile Lelong1,5, Emmanuelle Leize-Wagner 4, Mireille Chevallet 1,2 1: CEA, IRTSV, LBBSI, 38054 GRENOBLE, France. 2: CNRS, UMR 5092, Biochimie et Biophysique des Systèmes Intégrés, Grenoble France 3: Biomedical Proteomics Research Group, Central Clinical Chemistry Laboratory, Geneva University Hospitals, Geneva, Switzerland 4: CNRS, UMR 7177. Institut de Chime de Strasbourg, Strasbourg, France 5: Université Joseph Fourier, Grenoble France Correspondence : Thierry Rabilloud, iRTSV/LBBSI, UMR CNRS 5092, CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9 Tel (33)-4-38-78-32-12 Fax (33)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr Abstract: Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry( i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed. Keywords: electrophoresis, two-dimensional electrophoresis, isoelectric focusing, immobilized pH gradients, peptides, proteins, proteomics. Table of contents I. Introduction II. The principles at play III. How to use electrophoresis in a proteomics strategy III.A. -
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. -
Introduction to Capillary Electrophoresis
Contents About this handbook..................................................................................... ii Acronyms and symbols used ....................................................................... iii Capillary electrophoresis ...............................................................................1 Electrophoresis terminology ..........................................................................3 Electroosmosis ...............................................................................................4 Flow dynamics, efficiency, and resolution ....................................................6 Capillary diameter and Joule heating ............................................................9 Effects of voltage and temperature ..............................................................11 Modes of capillary electrophoresis ..............................................................12 Capillary zone electrophoresis ..........................................................12 Isoelectric focusing ...........................................................................18 Capillary gel electrophoresis ............................................................21 Isotachophoresis ...............................................................................26 Micellar electrokinetic capillary chromatography ............................28 Selecting the mode of electrophoresis .........................................................36 Approaches to methods development by CZE and MECC .........................37 -
Publications (1838-2000)
_________ NOTE: A bound copy of this bibliography is available without charge as long as the supply lasts. Send request to Dr. A. B. Chandler, Department of Pathology, BF-122, MCG or to >[email protected]< publications Dugas, L. A. Remarks on the pathology and treatment of bilious fever. Read before the Medical Society of Augusta. Southern Medical and Surgical Journal :–, . Dugas, L. A. Remarks on convulsions. Southern Medical and Surgical Journal :–, . Dugas, L. A. Operations on the eye. Southern Medical and Surgical Journal :–, . Dugas, L. A. Report on the ligamentum dentis. Southern Medical and Surgical Journal :–, . Dugas, L. A. Mortality in Augusta, during the years and . Southern Medical and Surgical Journal :–, . Dugas, L. A. Remarks on the pathology and treatment of convulsions. Southern Medical and Surgical Journal, New Series , no. :–, . Dugas, L. A. Extirpation of the mamma of a female in the mesmeric sleep, without any evidence of sensibility during the operation. Southern Medical and Surgical Journal, New Series :–, . Note: Authors’ names in bold type are pathology faculty and staff. medical college of georgia , cont’d. Dugas, L. A. Remarks on a lecture on mesmerism. Southern Medical and Surgical Journal, New Series :–, . Dugas, L. A. Extirpation of a schirrous tumor, the patient being in the mesmeric state, and evincing no sensibility whatever during the operation. Southern Medical and Surgical Journal, New Series :–, . Dugas, L. A. Extirpation of schirrous tumors from the mammary region and of an enlarged axillary gland—the patient having been rendered insensible by mesmerism. Southern Medical and Surgical Journal, New Series :–, . Dugas, L. A. Outlines of the pathological anatomy of the liver. -
Phos-Tagtm-Based Mobility Shift Detection of Phosphorylated Proteins
Phos-tag TM -based Mobility Shift Detection of Phosphorylated Proteins - Phosphate Affinity SDS-PAGE using Acrylamide-pendant Phos-tag TM – Ver. S2(2016/4) Introduction Phosphorylation is a fundamental covalent post-translational modification that regulates the function, localization, and binding specificity of target proteins. Methods for determining the phosphorylation status of proteins ( i.e., phosphoproteomics) are thus very important with respect to the evaluation of diverse biological and pathological processes. In 2002, Prof. Koike's group (Hiroshima University) reported that a dinuclear metal complex ( i.e. , 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) acts as a selective phosphate-binding tag molecule, Phos-tag TM in an aqueous solution at a neutral pH 2- (e.g. , Kd = 25 nM for phenyl phosphate dianion, Ph -OPO 3 ). Since then, various analytical methods for phosphoproteome research have been developed using Phos-tag TM derivatives. Here, we introduce two electrophoretic procedures for the simultaneous analysis of a phosphoprotein isoform and its non-phosphorylated counterpart: Method 1) Manganese(II)–Phos-tag TM SDS-PAGE using the Laemmli's buffer system, and Method 2) Zinc(II)–Phos-tag TM SDS-PAGE using a neutral pH buffer system. The methods provide characteristic separation patterns for phosphoprotein isoforms according to the number and/or site of phosphate group. Description of Acrylamide-pendant Phos-tag TM The acrylamide-pendant Phos-tag TM ligand (Phos-tag TM AAL-107) provides a phosphate affinity SDS-PAGE for mobility shift detection of phosphorylated proteins. This method requires only a general slab PAGE system. The products are supplied as 5mmol/L aqueous solution , which have no irritant effect on the skin. -
Supplementary Material
Supplementary Material Capillary Electrophoresis: Conceptual, Fundamentals and Instrumentation Electrophoresis was introduced by Tiselius in the beginning of the 1930s, through demonstration of the elegant method called mobile border, which demonstrated the partial separation of some protein constituents of blood serum. As a result of this pioneering work, the Nobel Award of 1948 was granted to Tiselius. Since then, electrophoresis has occupied a featured place among the methodologies used in biomolecules analyses. However, from the middle of the 1980s, through the implementation of capillaries techniques, electrophoresis has been developed from intensive manipulation to an automated format, deserving the status of a routine analytical technique [1,2]. Conceptually, CE is a separation technique that operates in liquid medium (aqueous or organic), which is based on the differentiated migration of neutral, ionic or ionisable compounds, when an electrical field in the order of kilovolts is applied tangentially to capillary tubes containing a background electrolyte (BGE) such as a buffer or a simple electrolyte solution [3,4]. When the electric field is applied, the compounds migrate with constant velocity and are separated into distinct zones, crossing the detector system that is conveniently positioned in a defined section of the capillary column. In general, the analyte separations are related to simultaneous contributions between electrophoretic mobility and electroosmotic flow (to be considered in the next paragraph). The electrophoretic mobility is an intrinsic characteristic of analytes and is dependent upon the charge of the molecule, the viscosity and the effective radius of the species. Overall, the electrophoretic mobility will be dependent of the size/charge relationship. Thus, if two ions are the same size, the one with the highest charge will move fastest because of the higher charge/size relationship. -
Chapter 2 15
Chapter 2 15 Chapter 2 Selectivity in Capillary Electrokinetic Separations [#] [#] This Chapter was published as a review in: T. de Boer, R.A. de Zeeuw, G.J. de Jong and K. Ensing. Electrophoresis, 20 (1999) 2989-3010 16 Chapter 2 Summary This review gives a survey of selectivity modes in capillary electrophoretic separations in pharmaceutical analysis and bioanalysis. Despite the high efficiencies of these separation techniques, good selectivity is required to allow quantitation or identification of a particular analyte. Selectivity in capillary electrophoresis is defined and described for different separation mechanisms, which are divided into two major areas: 1) capillary zone electrophoresis, and 2) electrokinetic chromatography. The first area describes aqueous (with or without organic modifiers) and non-aqueous modes. The second area discusses all capillary electrophoretic separation modes in which interaction with a (pseudo) stationary phase results in a change in migration rate of the analytes. These can be divided in micellar electrokinetic chromatography and capillary electrochromatography. The latter category can range from fully packed capillaries, via open tubular coated capillaries to the addition of microparticles with multiple or single binding sites. Furthermore, an attempt is made to differentiate between methods in which molecular recognition plays a predominant role and methods in which the selectivity depends on overall differences in physicochemical properties between the analytes. The calculation of the resolution for the different separation modes and the requirements for qualitative and quantitative analysis are discussed. It is anticipated that selectivity tuning can be easier in separation modes in which molecular recognition plays a role. However, sufficient attention needs to be paid to the efficiency of the system in that it not only affects resolution but also detectability of the analyte of interest. -
Separation and Detection of Large Phosphoproteins Using Phos-Tag SDS-PAGE
PROTOCOL Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE Eiji Kinoshita, Emiko Kinoshita-Kikuta & Tohru Koike Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima, Japan. Correspondence should be addressed to E.K. ([email protected]). Published online 24 September 2009; doi:10.1038/nprot.2009.154 We provide a standard phosphate-affinity SDS-PAGE (Mn2+–Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins (B150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted natureprotocols / migration bands on the improved Mn2+–Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of m o electrophoresis requires about 4 h in this protocol. c . e r u t a n INTRODUCTION . w Reversible phosphorylation is a key signaling mechanism for studies after electrophoresis, using radioactive compounds of w w 32 32 / modulating the functional properties of proteins involved in gene [g- P]-labeled ATP and [ P]-labeled orthophosphate. A meta- / : p t expression, cell adhesion, cell cycle, cell proliferation or cell bolic radiolabeling protocol has been successfully used in many t h 1 differentiation . -
High-Performance Capillary Electrophoresis Theory, Techniques, and Applications
High-Performance Capillary Electrophoresis Theory, Techniques, and Applications Edited by MORTEZA G. KHALEDI Department of Chemistry North Carolina State University Raleigh, North Carolina A Wiley-Interscience Publication JOHN WILEY & SONS, INC. New York / Chichester / Weinheim / Brisbane / Singapore / Toronto CONTENTS CONTRIBUTORS xix PREFACE xxiii CUMULATIVE LISTING OF VOLUMES IN SERIES xxvii PART I THEORY AND MODES OF HPCE CHAPTER 1 CAPILLARY ELECTROPHORESIS: OVERVIEW AND PERSPECTIVE 3 Barry L. Karger 1.1. Introduction 3 1.2. Modes of Operation 5 1.3. Capillary Electrophoresis-Mass Spectrometry 16 1.4. Electric Field Manipulation of Bulk Liquid Flow 18 1.5. Conclusions 22 List of Acronyms and Abbreviations 22 References 23 CHAPTER 2 THEORY OF CAPILLARY ZONE ELECTROPHORESIS 25 Ernst Kenndler 2.1. Capillary Zone Electrophoresis in the Absence of Electroosmotic Flow 27 2.2. Capillary Zone Electrophoresis in the Presence of Electroosmotic Flow 58 List of Symbols 70 List of Acronyms 73 References 73 v VI CONTENTS CHAPTER 3 MICELLAR ELECTROKINETIC CHROMATOGRAPHY 77 Morteza G. Khaledi 3.1. Introduction 77 3.2. Pseudostationary Phases 79 3.3. Migration in Micellar Electrokinetic Chromatography 83 3.4. Migration Parameters 84 3.5. Resolution 85 3.6. Structure-Retention Relationships in Micellar Electrokinetic Chromatography 87 3.7. Characterization of the Chemical Selectivity of Pseudostationary Phases 89 3.8. Effects of Chemical Composition of Micellar Solutions 95 3.9. Multiparameter Optimization 117 3.10. Conclusions and Future Trends 127 List of Acronyms 130 References 131 CHAPTER 4 BAND BROADENING IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY 141 Joe M. Davis 4.1. Introduction 142 4.2. Measurements of Efficiency 143 4.3. -
Affinity Electrophoresis May 11 (Wed.) 10:25 - 10:45
Affinity Electrophoresis May 11 (Wed.) 10:25 - 10:45 MICROHETEROGENEITY OF GLYCOPROTEINS T . C . B\2l g - Hansen The Protein Laboratory , University of Copenhagen , Denmark Affinity chromatography and electroimmunoprecipitati on can be combined to obtain analytical procedures with the following aims: To identify ligand- binding proteins , to characterize the binding with respect to structure and number of binding sites and the association constant, to quantify individual carbohydrat e f orms of microheterogeneolls glycoproteins , and to predict the results of preparative affinity separations . The following methods were developed to study glycoproteins with lectins , and they differ in the order in which proteins interact with lectins. In Method A the sample is incubated with l ectin before analysis by crossed immunoelectrophoresis . In Method B free lectin or immobilized l ectin is incorporated in an intermediate gel in crossed immunoelectrophoresis . In Method C free lectin or immobilized lectin is incorporated in the first dimension gel of crossed immunoelectrophoresis . By comparing with c ontrol experiments it is possible to identify and charac terize protein-lectin interactions by differences in electr opho retic and antigenic behavior of the protein under study. Protein lectin interaction can then be studied by comparison of the electr ophoretic and antigenic behavior of the proteins under study with the results of control experiments . Using these methods we have found that the microheterogeneity of serum glycoproteins is remarkably constant in normal indivi duals , but is changed characteristi cally in disease . Examples of quantitation of microheterogeneity forms of disease markers will be mentioned (eg. AFP , ferritin , AGP and other acute phase p r oteins ) and their biological significance will be discussed .