Download or read book Measuring the Size and Charge of Proteins Using Protein Charge Ladders Capillary Electrophoresis and Electrokinetic Models of Colloids written by Jeffrey D. Carbeck and published by . This book was released on 2001 with total page 4 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Capillary Electrophoresis of Proteins and Peptides written by Mark A. Strege and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 340 pages. Available in PDF, EPUB and Kindle. Book excerpt: Throughout the more than 20 years that have followed the beginnings of capillary electrophoresis (CE), its application to the analysis of proteins and peptides has continued to be reliable, versatile, and productive. Over time, CE has matured to become a superb complement to HLPC, and in many cases has also evolved as an automated and quantitative replacement for conventional slab gel electrophoresis methods such as SDS-PAGE and isoelectric focusing. Within Capillary Electrophoresis of Proteins and Peptides, we have assembled contributions from researchers who are applying state-of-the-art CE for protein and peptide analysis, including topics that we believe are of great potential both in the present and for the future. In comparison to traditional separation methods, CE represents a miniaturized analysis technique (especially in its microchip-based format) that is highly dependent upon the basic fundamentals of effective sample recovery and high sensitivity detection. With these issues in mind, Chapters 1–4 describe recently developed approaches for both capillary coatings and analyte detection via laser-induced fluorescence. Since the discipline of biotechnology has established itself as a primary platform for the application of CE to the analysis of proteins and peptides, Chapters 5–7 demonstrate a variety of examples of the specific techniques that have been applied for the development of biopharmaceuticals and their commercialization. The methods covered here include also the analysis of oligosaccharides from glycoproteins.
Download or read book Structure in Protein Chemistry written by Jack Kyte and published by Garland Science. This book was released on 2006-11-01 with total page 1505 pages. Available in PDF, EPUB and Kindle. Book excerpt: The second edition of Structure in Protein Chemistry showcases the latest developments and innovations in the field of protein structure analysis and prediction. The book begins by explaining how proteins are purified and describes methods for elucidating their sequences of amino acids and defining their posttranslational modifications. Comprehensive explanations of crystallography and of noncovalent forces-ionic interactions, hydrogen bonding, and the hydrophobic effect-act as a prelude to an exhaustive description of the atomic details of the structures of proteins. The resulting understanding of protein molecular structure forms the basis for discussions of the evolution of proteins, the symmetry of the oligomeric associations that produce them, and the chemical, mathematical, and physical basis of the techniques used to study their structures. The latter include image reconstruction, nuclear magnetic resonance spectroscopy, proton exchange, optical spectroscopy, electrophoresis, covalent cross-linking, chemical modification, immunochemistry, hydrodynamics, and the scattering of light, X-radiation, and neutrons. These procedures are applied to study the folding of polypeptides and the assembly of oligomers. Biological membranes and their proteins are also discussed. Structure in Protein Chemistry, Second Edition, bridges the gap between introductory biophysical chemistry courses and research literature. It serves as a comprehensive textbook for advanced undergraduates and graduate students in biochemistry, biophysics, and structural and molecular biology. Professionals engaged in chemical, biochemical, and molecular biological research will find it a useful reference.
Download or read book Topical Conference Proceedings of the 2001 Bioinformatics and Genomics Symposium and the 2001 Annual Meeting of the American Electrophoresis Society written by and published by . This book was released on 2001 with total page 200 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Charge Ladder Measurements of Protein Charge and Size Under Simulated Intracellular Conditions written by Shaun W. Shelton and published by . This book was released on 2007 with total page 160 pages. Available in PDF, EPUB and Kindle. Book excerpt: A protein charge ladder is a series of protein derivatives with incrementally differing charge (AZ), often made by acetylation or amidation of charged side chains. The electrophoretic mobility (//eP) of each "rung" of the charge ladder is measured by capillary zone electrophoresis (CZE). The net charge (ZCE) and hydrodynamic radius (i?h) of the unmodified protein can be determined from a plot of /J^V versus AZ. Simulating the intracellular environment ( -200 mM ionic strength and 50-150 mg/mL total protein) in CZE brings additional challenges. In conventional CZE with 50-75 um inner diameter capillaries and electric fields of -400 V/cm, the ionic strength cannot greatly exceed -25 mM without causing excessive band broadening due to Joule heating. We have been able to accommodate ionic strengths up to -220 mM at 400 V/cm using 20 um capillaries. Since UV absorbance detection is not practicable in these capillaries due to the reduced optical path length and greatly increased stray light, we use laser-induced fluorescence (LIF) detection. LIF also allows us to detect a fluorescently labeled protein of interest in the presence of high concentrations of unlabeled proteins. Charge ladders of a-lactalbumin have been produced by modification of Lys residues with fluorescein isothiocyanate (FITC), which gives a nominal AZof-2. The estimated electrostatic zeta potential (Q of the unmodified protein is -26 mV, which already exceeds the 25 mV limit of the Debye-Hiickel approximation that is used to interpret the charge ladder data. The FITC-modified rungs have even more negative Rvalues. Our procedure overestimates the net charge if the rungs of the charge ladder do not fall in the linear Debye-Hiickel regime. The estimated ZCE increases unrealistically at higher ionic strengths, approaching the total number of negatively charged groups on the protein. To reduce the Rvalues of our charge variants, we decoupled the charge ladder from labeling with FITC. The protein is lightly labeled first to prevent multiple FITC labeling. The charge ladder is then formed by amidating Asp and Gly residues (AZ = +1). The following parameters were estimated for a-lactalbumin at pH 8.3 and low ionic strength: ZCE = -7.1, i?h = 2.5 ± 0.7 nm, and C, = -21.6 mV. LIF was then used to detect the lightly labeled charge ladder under conditions varying in I, up to 220 mM. As I increased, ZCE increased from -7.2 ± O.lto -8.8 ± 0.1, did not show any real trend, and C, decreased from -23 to -13 mV. To simulate macromolecularly crowded conditions, BSA was used as the crowding agent at a concentration of 75 mg/mL. The charge ladder was then run under crowded conditions at low ionic strength, and values of ZCE = -4.8, Rh = 2.9 ± 0.7 nm, and C, = -12 mV were estimated for unmodified protein. At conditions of high ionic strength (100 or 200 mM) ZCE became more negative ( -4.8 to -6.6), varied between 2.9-2.6 nm and C, decreased from -12.8 to -7.8 mV. The viscosity of the solution is 1.6 times higher than in water based solutions and this caused severe losses in the resolution of adjacent rungs of the charge ladder. A loss in resolution is a direct of effect to the value of the slope being so small. Affinity capillary electrophoresis was used in studying the binding behavior between calcium and a-lactalbumin. Lightly labeled and amidated a-lactalbumin was run in Ca2+ concentrations of 3.5 and 10 mM CaC^. The charge ladder tended to "collapse" at these high Ca' concentrations. We switched to an acetylated ladder to reduce the possibility of modifying any residues specific for binding calcium. At lower Ca2+ concentrations (100 uM) the charge ladders were resolved. ZCE was estimated to be -6.1 at 0 uM Ca2+ as compared to -5.8 at 100 uM Ca2+. Rh was fairly constant between 2.5 and 2.4 nm. A small change in ZCE and suggests that binding between the charge ladder and Ca2+ occurred. However, there was no noticeable change in the Rh as expected.
Download or read book Index Medicus written by and published by . This book was released on 2001 with total page 1736 pages. Available in PDF, EPUB and Kindle. Book excerpt: Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.
Download or read book Environmental Colloids and Particles written by Kevin J. Wilkinson and published by John Wiley & Sons. This book was released on 2007-01-30 with total page 702 pages. Available in PDF, EPUB and Kindle. Book excerpt: This text presents the current knowledge of environmental colloids and includes reviews of the current understanding of structure, role and behaviour of environmental colloids and particles, whilst focussing directly on aquatic systems and soils. In addition, there is substantial critical assessment of the techniques employed for the sampling, size fractionation and characterisation of colloids and particles. Chemical, physical and biological processes and interactions involving colloids are described, and particular attention is paid to quantitative approaches that take account of particle heterogeneity and polydispersity. Presents critical reviews of the state-of-the-art knowledge of environmental colloids Critical assessment of techniques employed for the sampling, size fractionation and characterisation of colloids and particles are given Theoretical and experimental aspects of the methods as well as the required developments and possible recommendations are discussed Each chapter gives a brief introduction general enough for the non-specialist Written by a internationally recognized group of contributors
Download or read book Biophysico Chemical Processes Involving Natural Nonliving Organic Matter in Environmental Systems written by Nicola Senesi and published by John Wiley & Sons. This book was released on 2009-07-23 with total page 905 pages. Available in PDF, EPUB and Kindle. Book excerpt: An up-to-date resource on natural nonliving organic matter Bringing together world-renowned researchers to explore natural nonliving organic matter (NOM) and its chemical, biological, and ecological importance, Biophysico-Chemical Processes Involving Natural Nonliving Organic Matter in Environmental Systems offers an integrated view of the dynamics and processes of NOM. This multidisciplinary approach allows for a comprehensive treatment encompassing all the formation processes, properties, reactions, environments, and analytical techniques associated with the latest research on NOM. After briefly outlining the historical background, current ideas, and future prospects of the study of NOM, the coverage examines: The formation mechanisms of humic substances Organo-clay complexes The effects of organic matter amendment Black carbon in the environment Carbon sequestration and dynamics in soil Biological activities of humic substances Dissolved organic matter Humic substances in the rhizosphere Marine organic matter Organic matter in atmospheric particles In addition to the above topics, the coverage includes such relevant analytical techniques as separation technology; analytical pyrolysis and soft-ionization mass spectrometry; nuclear magnetic resonance; EPR, FTIR, Raman, UV-visible adsorption, fluorescence, and X-ray spectroscopies; and thermal analysis. Hundreds of illustrations and photographs further illuminate the various chapters. An essential resource for both students and professionals in environmental science, environmental engineering, water science, soil science, geology, and environmental chemistry, Biophysico-Chemical Processes Involving Natural Nonliving Organic Matter in Environmental Systems provides a unique combination of the latest discoveries, developments, and future prospects in this field.
Download or read book Capillary Electrophoresis of Proteins written by Tim Wehr and published by CRC Press. This book was released on 1998-10-20 with total page 301 pages. Available in PDF, EPUB and Kindle. Book excerpt: "Provides practical information on the application of capillary electrophoresis (CE) to protein analysis, with an emphasis on developing and optimizing CE techniques in the laboratory. Includes separation methods bases on mass, charge, isoelectric point, molecular sieving, and affinity interactions."
Download or read book Protein Charge Ladders and Capillary Electrophoresis written by Jeffrey D. Carbeck and published by . This book was released on 1998 with total page 7 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Journal of Chromatography written by and published by . This book was released on 2001 with total page 604 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Capillary Electrophoresis of Proteins and Peptides written by Mark A. Strege and published by Humana. This book was released on 2010-11-10 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Throughout the more than 20 years that have followed the beginnings of capillary electrophoresis (CE), its application to the analysis of proteins and peptides has continued to be reliable, versatile, and productive. Over time, CE has matured to become a superb complement to HLPC, and in many cases has also evolved as an automated and quantitative replacement for conventional slab gel electrophoresis methods such as SDS-PAGE and isoelectric focusing. Within Capillary Electrophoresis of Proteins and Peptides, we have assembled contributions from researchers who are applying state-of-the-art CE for protein and peptide analysis, including topics that we believe are of great potential both in the present and for the future. In comparison to traditional separation methods, CE represents a miniaturized analysis technique (especially in its microchip-based format) that is highly dependent upon the basic fundamentals of effective sample recovery and high sensitivity detection. With these issues in mind, Chapters 1–4 describe recently developed approaches for both capillary coatings and analyte detection via laser-induced fluorescence. Since the discipline of biotechnology has established itself as a primary platform for the application of CE to the analysis of proteins and peptides, Chapters 5–7 demonstrate a variety of examples of the specific techniques that have been applied for the development of biopharmaceuticals and their commercialization. The methods covered here include also the analysis of oligosaccharides from glycoproteins.
Download or read book Role of Electrostatics in Protein Stability Measured by Capillary Electrophoresis and Protein Charge Ladders written by Jeffrey D. Carbeck and published by . This book was released on 2001 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Charge Information Based on Electrophoretic Mobility for Multidimensional Protein Analysis written by Rania Ameen Dumarieh and published by . This book was released on 2006 with total page 140 pages. Available in PDF, EPUB and Kindle. Book excerpt: Two-dimensional separation techniques are widely used because of their large peak capacity (their ability to resolve a large number of sample components). Two-dimensional gel electrophoresis (2DGE) is one of the most popular techniques for proteome analysis mainly because of its large peak capacity and ease of interpretation. One of its major limitations, however, is that it is time consuming. Jorgenson's group[superscript]3,4 has presented size exclusion chromatography-capillary zone electrophoresis (SEC-CZE) as a potential alternative for 2DGE. One of the major potential advantages associated with SEC-CZE is speed; sensitivity is secondary. However, the peak capacity of the technique, which is directly related to the orthogonality between the two dimensions, has not been directly reported. We investigated the issue of orthogonality between net charge and size theoretically by calculating theoretical charge values for 4290 proteins expressed in E. coli, and found that net charge begins to correlate with size as pH increases, due to the correlation between the absolute number of ionizable groups on a protein and protein size. Because of the difference between theoretical and actual net charge, we used CZE and charge ladders to measure net charge for a collection of model proteins as a function of pH. Although enolase (baker's yeast, pI = 6.17, MW = 47 kDa) is more basic than [alpha]-lactalbumin (bovine milk, pI = 4.6, MW = 14.2 kDa), it has net charge that is similar to that of [alpha]-lactalbumin at intermediate pH values, but it has higher net charge at higher pH values. For proteins of similar pI but varying size, such as ubiquitin (bovine red blood cells, pI = 7.0, MW = 8.6 kDa), myoglobin (horse skeletal muscle, pI = 6.8, MW = 17.6 kDa) and creatine phosphokinase (rabbit muscle, pI = 7.1, MW = 86.6 kDa), there is correlation between net charge and size even at intermediate pH values. For SEC-CZE to be used as an alternative to 2DGE, the information obtained from SEC-CZE should be interpretable. The second main question this research is trying to answer is: can we combine size data obtained from SEC with electrophoretic mobility data obtained from CZE to obtain charge information? Attempts at obtaining size measurements from fabricated SEC capillary columns will be discussed. Also, some of the limitations of using electrophoretic mobility data to determine net charge, such as high protein surface potentials, will be illustrated using the amidated and acetylated charge ladders of a model protein that is expected to have high surface potential (soybean trypsin inhibitor, pI = 4.6, MW = 20.1 kDa). For a protein with high surface potential, using simple equations of electrophoresis to estimate net charge from electrophoretic mobility ([mu][subscript]ep) and hydrodynamic radius (R[subscript]h) data, underestimates the net charge of the protein mainly because of ion relaxation effects. It is advisable to run SEC-CZE at intermediate pH because of the better orthogonality between net charge and size in that pH region. Also, proteins are more likely to have low surface potentials in that pH region, and, as a result, more reliable estimates of Z[subscript]CE can be obtained by combining R[subscript]h and [mu][subscript]ep measurements. Because of the low overall peak capacity of SEC-CZE and because of the difficulty of interpreting electrophoretic mobility even if converted to a charge estimate, it is unlikely that SEC-CZE will ever become a widely used alternative to 2DGE.
Download or read book Journal written by American Chemical Society and published by . This book was released on 2001 with total page 1320 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Proteins and the Theory of Colloidal Behavior written by Jacques Loeb and published by . This book was released on 1924 with total page 410 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Capillary Electrophoresis of Proteins and Peptides written by Nguyet Thuy Tran and published by Humana. This book was released on 2018-05-30 with total page 234 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a comprehensive survey of recent developments and applications of high performance capillary electrophoresis in the field of protein and peptide analysis with a distinct focus on the analysis of intact proteins. With practical detail, the contents cover different modes of capillary electrophoresis (CE) useful for protein and peptide analysis, CZE, CIEF, ACE, CGE, and different types of application such as the quality control of therapeutic proteins and monoclonal antibodies, clinical analyses of chemokines in tissues, qualitative and quantitative analysis of vaccine proteins, and determination of binding constants in complexes involving peptides or proteins. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and exhaustive, Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols serves both beginners and experts with a collection of the current and most active topics in this vital field of study.
