Biological Sciences
Gel electrophoresis
Gel electrophoresis is a technique used to separate and analyze DNA, RNA, or proteins based on their size and charge. The molecules are loaded into a gel matrix and then subjected to an electric field, causing them to migrate through the gel. This allows for the visualization and comparison of different molecules based on their mobility.
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eBook - PDFBasic Bioscience Laboratory Techniques
A Pocket Guide
- Philip L.R. Bonner, Alan J. Hargreaves(Authors)
- 2022(Publication Date)
- Wiley-Blackwell(Publisher)
Basic Bioscience Laboratory Techniques: A Pocket Guide, Second Edition. Philip L.R. Bonner and Alan J. Hargreaves. © 2022 John Wiley & Sons Ltd. Published 2022 by John Wiley & Sons Ltd. 6 97 6.1 General Introduction Electrophoresis is a technique that is used to separate charged molecules in an inert support matrix under the influence of an electric field. The most popular inert support for the electrophoresis of proteins is polyacrylamide (see Chapter 6, Section 6.2) and for the electrophoresis of nucleic acids, it is agarose (see Chapter 6, Section 6.4). When an electric field is applied, different biological molecules will move at different rates and the components of a mixture can be separated from each other. Electrophoresis has numerous applications in bioscience research, including the separation of proteins and nucleic acids, which will be the focus of this chapter. It is typically used in the analysis of complex mixtures (e.g. cell and tissue extracts) or in the monitoring of purity of isolated macromolecules (e.g. proteins, DNA, and so on). The electrophoretic separation of any molecule depends on opposing forces of propulsion and resistance, as outlined below: Propulsion is driven by: a. Charge – negatively charged molecules (anions) migrate towards the anode (+) and positively charged molecules (cations) migrate towards the cathode (−). b. Field strength – higher voltage promotes faster migration up to a certain limit, when overheating can occur, which will distort the electrophoretic migration pattern and could damage the resultant gel. Resistance to migration is dependent on: a. The size and shape of the biomolecules being separated – larger molecules migrate through a solution or matrix at a slower rate than smaller (lower relative mass) molecules as they experience greater frictional drag. Similarly, bulky molecules will experience more resistance than molecules with a linear shape.
eBook - ePubProteomic Profiling and Analytical Chemistry
The Crossroads
- Pawel Ciborowski, Jerzy Silberring(Authors)
- 2016(Publication Date)
- Elsevier(Publisher)
7.2.5 Pros and Cons of Two-Dimensional Gel electrophoresis 1357.2.6 Quantitation of Protein Using Two-Dimensional Gels 1367.2.7 Difference Gel electrophoresis 1397.2.8 Fluorescent Dyes Used in Difference Gel electrophoresis 1407.2.9 Internal Standard 1417.2.10 Pros and Cons of Difference Gel electrophoresis 142References 1427.1 Fundamentals of Gel electrophoresisA. Drabik AGH University of Science and Technology, Krakow, PolandJ. Silberring AGH University of Science and Technology, Krakow, Poland Polish Academy of Sciences, Zabrze, Poland7.1.1. Introduction
Electrophoretic separation is based on the migration of unbalanced charged molecules in an electric field and is the most frequently used dispensation method in the study of proteins and nucleic acids. It is widely used in biochemistry, molecular biology, pharmacology, criminal medicine, diagnostics and food quality control. Electrophoresis can be used for macromolecule isolation in complex biological systems, as well as a tool for determining molecular weight (MW) and detecting structural and charge-state modifications. It can also be applied as a sample purity control, as well as a discovery tool for proteins, nucleic acids and large peptides.The main premise of electrophoretic separation is application of an electric field that forces molecules to move through gel pores, separating them based on their MW and total particle charge. Large-molecular-weight molecules are slowed down on the basis of gel pore size (Tables 7.1.1 and 7.1.2 ); more specifically, larger-molecular-weight molecules are “trapped” in regions of the gel with a higher percent concentration [1]
eBook - ePubProteomic Profiling and Analytical Chemistry
The Crossroads
- Pawel Ciborowski, Jerzy Silberring(Authors)
- 2012(Publication Date)
- Elsevier(Publisher)
Electrophoretic separation is based on the migration of unbalanced charged molecules in an electric field and is the most frequently used dispensation method in the study of proteins and nucleic acids. It is widely used in biochemistry, molecular biology, pharmacology, criminal medicine, diagnostics, and food quality control. Electrophoresis can be used for macromolecule isolation in complex biological systems, as well as a tool for determining molecular weight and detecting structural and charge state modifications. It can also be applied as a sample purity control, as well as a discovery tool for proteins, nucleic acids, and large peptides.The main premise of electrophoretic separation is application of an electric field that forces molecules to move through gel pores, separating them based on their molecular weight and total particle charge. Large molecular weight molecules are slowed down on the basis of gel pore size (Tables 6.1.1 and 6.1.2 )—more specifically, larger molecular weight molecules are “trapped” in regions of the gel with a higher percent concentration. The migration ratio is a constant value that is directly proportional to the electric current, shape, and size of separated species, hydrophobicity, ionic strength, viscosity, and temperature as defined here:Table 6.1.1 Molecular separation range in function of agarose gel concentrationAgarose concentration (g/100 ml) Molecule size (kDa) 0.3 5–60 0.6 1–20 0.7 0.8–10 0.9 0.5–7 1.2 0.4–6 1.5 0.2–3 2 0.1–2 Table 6.1.2 Acrylamide concentration correlation with separated species molecular weightwhere μ is electrophoretic mobility, V is migration speed, E is electric field strength, Z is total molecular charge, is friction coefficient, η is viscosity, and r is molecule radius.Application of a specially designed electrophoretic power supply enables the user to keep a constant value of a selected parameter: voltage, current, or power. During the separation process, electrolyte resistance is reduced by temperature increases, while a reduction in the number of ions and their arrangement order in the gel can increase the resistance. When the temperature is elevated, the separation time is extended, which can cause molecules to diffuse, thereby decreasing the resolving power.
- Atul Bhargava, Shilpi Srivastava(Authors)
- 2017(Publication Date)
- CRC Press(Publisher)
5 ElectrophoresisA Conceptual UnderstandingGurjeet Kaur and Vineet Awasthi CONTENTS 5.1 Introduction 5.2 Principle 5.3 Agarose Gel electrophoresis 5.4 Pulsed-Field Gel electrophoresis 5.5 Polyacrylamide Gel electrophoresis 5.6 Native PAGE 5.7 Sodium Dodecyl Sulfate–Polyacrylamide Gel electrophoresis 5.8 Gradient Gels 5.9 Isoelectric Focusing 5.10 Two-Dimensional Electrophoresis 5.11 Capillary Electrophoresis Bibliography 5.1 INTRODUCTIONArne Tiselius in 1931 developed a new instrument called Tiselius apparatus for moving boundary electrophoresis for separation and chemical analysis of the charged particles when subjected to electric field. The work was supported by the Rockefeller Foundation. With the evolution, the technique became more sensitive and effective and was known as zone electrophoresis. Later, the support media used was in the form of gels or filter paper, thus making electrophoresis and its related techniques an earnest tool in the bioscientific laboratories. With the advancement in the newer versions of techniques for the study of biomolecules, electrophoresis has never lost its momentum of usage in the separation of macromolecules. Electrophoresis is used as an individual as well as a supporting technique in many ways.5.2 PRINCIPLEElectrophoresis is based on the principle of movement of charged particles through a solution under the influence of electric field. It is the electromotive force that is used to move the molecule through the support medium. Every macromolecule possesses some net charge (q). When the electric field is applied on the molecules, the electric field strength, E, is experienced by the molecule. The molecules move toward the oppositely charged electrodes. If the molecule has net positive charge (cations), then it migrates toward cathode (cationic [negative] electrode); similarly, if the molecule has net negative charge, it migrates toward anode (anionic [positive] electrode). The migration of the molecule depends on the driving force and the resisting force experienced by the molecule. The support medium is known to suppress the thermal convection resulting due to the electric field applied, thereby retarding the movement of the molecule through the sieves formed by the support media. It is also clear that the sieves formed allow the molecule to stay at the point of separation and are ultimately stained to be visualized. The migration through the sieves of the support media allows the smaller molecules to migrate faster in comparison with the larger molecules. Thus, it is clear that the separation with the help of electrophoresis is based on size and charge possessed by the molecule. The support media commonly referred to as gels are prepared in certain percentage. The change in percentage is responsible for the pore size of the gel. The size of the molecule to be separated suggests the percentage of the gel formed for conducting electrophoresis. Higher the percentage of the gel, the smaller will be the pore size (inversely proportional). It can also be framed that bigger the molecules to be separated, smaller will be the percentage of the gel (directly proportional).
eBook - PDF- George D. J. Phillies(Author)
- 2011(Publication Date)
- Cambridge University Press(Publisher)
3 Electrophoresis 3.1 Introduction The early electrophoresis experiments of Tiselius, first published in 1930, examined the motions of proteins in bulk solution as driven by an applied electrical field(1). In the original method, a mixture of proteins began at a fixed location. Under the influence of the field, different protein species migrated through solution at differ- ent speeds. In time, the separable species moved to distinct locations (“bands”). Electrophoresis is now a primary technique for biological separations(2, 3). Two improvements were critical to establishing the central importance of electrophore- sis in biochemistry: First, thin cells and capillary tubes replaced bulk solutions. Second, gels and polymer solutions replaced the simple liquids used by Tiselius as support media. These two improvements greatly increased the resolution of an electrophoretic apparatus. Electrophoresis in true gels is a long-established exper- imental method. The use of polymer solutions as support media is more recent. An early motivation for their use was the suppression of convection, but electrophoretic media that enhance selectivity via physical or chemical interaction with migrating species are now an important biochemical tool. Electrophoresis and sedimentation have a fundamental similarity: in each method, one observes how particular molecules move when an external force is applied to them. In sedimentation, the enhancement of buoyant forces by the ultracentrifuge causes macromolecules to settle or rise. In electrophoresis, the applied electrical field causes charged macromolecules to migrate. The experi- mental observable is the drift velocity of the probe as one changes the molecular weight and concentration of the matrix, the size or shape of the probe, or the strength of the external force. Historically, sedimentation and electrophoresis have both been viewed as methods for studying the properties of the migrating species.
Available until 25 Jan |Learn more- Richard Li(Author)
- 2015(Publication Date)
- CRC Press(Publisher)
159 8 DNA Electrophoresis It is necessary to separate various sizes of DNA fragments so that the DNA fragment in ques- tion can be identified and analyzed. This can be achieved by electrophoresis, a process in which fragments are separated based on the migration of charged macromolecules in an electric field. 8.1 Basic Principles DNA is a negatively charged molecule in an aqueous environment, with the phosphate groups of DNA nucleotides carrying negative charges. DNA molecules migrate from the negative elec- trode (cathode) toward the positive electrode (anode) in an electric field during electrophoresis. The electrical potential, a measure of the work required to move a charged molecule in an electric field, is the force responsible for moving the charged macromolecules during electrophoresis. The electrophoretic mobility of macromolecules is primarily determined by their charge-to- mass ratios and their shapes. However, because the phosphate group of every DNA nucleotide carries a negative charge, the charge-to-mass ratio of DNA molecules is almost the same even if the length of the DNA fragment varies. Additionally, forensic DNA testing usually analyzes linear DNA molecules such as double-stranded linear DNA for variable number tandem repeat (VNTR) analysis (Chapter 19) and single-stranded DNA for short tandem repeat (STR) analysis (Chapter 20). The shapes of linear DNA molecules are similar. Therefore, the electrophoretic separation of different-sized DNA fragments, through a series of pores of the supporting matrix in which the fragments travel, is based more on their sizes than their shapes. It is obviously easier for smaller molecules to migrate through the pores of a supporting matrix than larger molecules; this is why smaller molecules migrate faster through the matrix. Hence, the electrophoretic mobility increases as the size of the DNA molecule decreases.
- Alexander J. Ninfa, David P. Ballou, Marilee Benore(Authors)
- 2015(Publication Date)
- Wiley(Publisher)
161 Gel electrophoresis of Proteins 6.1 Process of Electrophoresis 6.2 Polyacrylamide Gels 6.3 SDS-Polyacrylamide Gel electrophoresis (SDS-PAGE) of Proteins 6.4 Detection of Proteins in SDS-Polyacrylamide Gels 6.5 Applications of SDS-PAGE Experiments 6-1 and 6-2 Reagents Needed for Chapter 6 6.1 PROCESS OF ELECTROPHORESIS When an electric field is placed across a solution containing ions, a current develops in which anions (negatively charged species) move towards the anode and cations (positively charged species) move towards the cathode. The moving ions constitute the current that flows between the electrodes. A solution that contains few ions (e.g., pure distilled water or benzene) would have very high resistance and would carry very little current. Mobility is the term describing how easily the ions move in an electric field. Electrophoresis is the process by which charged molecules are separated in an electric field due to their differential mobilities. Factors affecting the mobility of a molecule in an electric field are the charge of the molecule q, and the voltage gradient of the electric field E, which together result in the force to move the ions, and the frictional resistance of the supporting medium (f), which impedes their movement. Mobility is defined as the rate of migration traveled with a voltage gradient of 1V/cm, and has units of cm 2 /sec/V. For practical reasons, one usually determines relative mobilities of proteins, R f , which is the ratio of the distance a protein migrates to that of a small anionic dye (the tracking 6 Fundamental Laboratory Approaches for Biochemistry and Biotechnology 162 dye). The high charge-to-mass ratio of the dye causes the dye to migrate very near to the electrophoretic front and ahead of the proteins.
eBook - PDF- M.R. El-Gewely(Author)
- 1997(Publication Date)
- Elsevier Science(Publisher)
This can be achieved by following optimized protocols and by standardizing all steps: from the preparation of samples, buffers and gels to the electrophoresis conditions and the manipulation of gels. Preparation of large stocks of master solutions and minimizing manual handling are two basic points for an efficient daily routine. Applications in different fields In principle, almost any pool of DNA fragments can be efficiently separated by two-dimensional electrophoresis, which generates a highly informative pattern of spots under optimized conditions. Genomic DNA isolated from plants, ani- mals, bacteria or viruses can be analyzed after enzymatic digestion or amplified by polymerase chain reaction using specific or arbitrary primers [65-671. The Fig. 8. Gel analysis and reproducibility, Comparison of reproducibility has been performed in several inter- and intralaboratory experiments. A simplified example of computerized analysis of the spot patterns is shown. The gels are representative of different electrophoresis runs or of different gels with- in the same run (up to 10 gels). For this experiment the semiautomated prototype (Fig. 4) and the Ingenyvision image analysis system described in the text were used [a]. After the calibration step (Fig. 6), computer analysis makes it possible to compare up to 16 gels in parallel. On the upper left is a reference database containing different spots corresponding to different restriction fragments (phage lambda DNA). 13 possibility to generate a fingerprint on the basis of both the size and the sequence of the obtained fragments, represents a powerll tool for genetic typing of micro- organisms, animals and plants, and can contribute to medical genetics and foren- sic medicine. The complex spot patterns obtained after hybridization with highly polymorphic multilocus probes represent displays for analysis of genomic instability, assessment of mutation rates and possible association studies.
eBook - PDFIntegrated Microfabricated Biodevices
Advanced Technologies for Genomics, Drug Discovery, Bioanalysis, and Clinical Diagnostics
- Michael J. Heller, Andras Guttman, Michael J. Heller, Andras Guttman(Authors)
- 2002(Publication Date)
- CRC Press(Publisher)
7 Electric-Field-Mediated Separation of DNA Fragments on Planar Microgels Andras Guttman Torrey Mesa Research Institute, San Diego, California 7.1 INTRODUCTION Throughput is one of the greatest single factors impacting the cost- effectiveness of large-scale genetic mapping, mutation (SNP) de- tection, or PCR product analysis by using conventional agarose or polyacrylamide Gel electrophoresis systems [1]. Despite numerous refinements in electrophoresis techniques over the past decade, the process is still not efficient enough and hardly automated. The time required to preparing, loading, and separating DNA fragments using conventional Gel electrophoresis, and staining and exposure times are all added up. Although several attempts have been made to automate this technology [2], DNA fragment analysis in most laboratories is still done in a very conventional way: using slab Gel electrophoresis separa- tion with ethidium bromide staining [3], or more recently with novel, higher-sensitivity fluorophores [4]. The introduction of fluorescent dyes provided the possibility of employing regular or electronic (CCD) cam- eras to take pictures of transilluminated gels for data evaluation and archiving [5]. By all means, the existing technology of separating DNA 165 DOI: 10.1201/9780203908006-7 166 Guttman fragments using Gel electrophoresis is a task that requires multiple labor-intensive steps, such as gel casting, sample loading, staining and imaging/documentation/data evaluation. These tasks are not readily in- tegrated and automated for high-throughput applications. Some of the large, automated DNA sequencing systems have recently been reported to accommodate genotyping and STR profiling [6] using crosslinked polyacrylamide gels and fluorescently labeled primers; however, the configuration of these devices does not accommodate the use of agarose gels as separation medium.
eBook - PDFElectrophoresis
Theory and Practice
- Budin Michov(Author)
- 2020(Publication Date)
- De Gruyter(Publisher)
Electrophoresis , 2009, 30, 2014 – 2024. [23] Lucotte G, Baneyx F. Introduction to Molecular Cloning Techniques . Wiley-Blackwell, 1993, 32. [24] Bahga SS, Bercovici M, Santiago JG. Electrophoresis , 2010, 31, 910 – 919. [25] Michov BM. Electrochimica Acta , 2013, 108, 79 – 85. [26] Aaij C, Borst P. Biochim Biophys Acta , 1972, 269, 192 – 200. [27] Voet D, Voet JG. Biochemistry , 2nd edn. John Wiley & Sons, 1995, 877 – 878. [28] Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Ehrlich HA, Arnheim N. Science , 1985, 230, 1350 – 1354. [29] Haas H, Budowle B, Weiler G. Electrophoresis , 1994, 15, 153 – 158. [30] Michov BM. Protein separation by SDS electrophoresis in a homogeneous gel using a TRIS-formate-taurinate buffer system and suitable homogeneous electrophoresis plates (Proteintrennung durch SDS-Elektrophorese in einem homogenen Gel unter Verwendung eines TRIS-Formiat-Taurinat-Puffersystems und dafür geeignete homogene Elektrophoreseplatten). German Patent 4127546, 1991. [31] Allen RC, Budowle B. Gel electrophoresis of Proteins and Nucleic Acids . Walter de Gruyter, Berlin ‒ New York, 1994, 262. [32] Ansorge W. In Stathakos D (ed.), Electrophoresis ´82 . Walter de Gruyter, Berlin ‒ New York, 1983, 235. [33] Mayrand PE, Corcoran KP, Ziegle JS, Robertson JM, Hoff LB, Kronick MN. Appl Theoret Electrophoresis , 1992, 3, 1. [34] Robertson J, Ziegle J, Kronick M, Madden D, Budowle B. In Burke T, Dolt G, Jeffries AJ, Wolf R (eds.), Fingerprinting. Approaches and Applications . Birkhäuser, Basel, 1991, 391. [35] Rüger R, Höltke HJ, Reischl U, Sagner G, Kessler C. J Clin Chem Clin Biochem , 1990, 28, 566. [36] PCR Clean-up Gel Extraction , User Manual. Macherey-Nagel, 2017, Rev. 04, 9. [37] Szoke M, Sasvari-Szekely M, Barta C, Guttman A. Electrophoresis , 1999, 20, 497 – 501. [38] Maurer HR, Dati FA. Anal Biochem , 1972, 46, 19 – 32. [39] Guttman A, Barta C, Szoke M, Sasvari-Szekely M, Kalasz H.
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