Capillary Electrophoresis with Post-column Laser-induced Fluorescence Detection of Proteins PDF Download
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Languages : en
Pages : 42
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Book Description
Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is one of the alternative methods being developed for fast and sensitive assays of proteins and peptides. This report summarizes CE-LIF instrument development, efforts at labelling proteins, tests conducted to prove the applicability of CE-LIF to the analysis of samples, and results of a comparison of CE-LIF with some other techniques. Appendices include an instrument operating procedure and a paper that summarizes the experimental details of the protein labelling procedure and a CE-LIF technique for sub-nanomolar assay of proteins.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 42
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Book Description
Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is one of the alternative methods being developed for fast and sensitive assays of proteins and peptides. This report summarizes CE-LIF instrument development, efforts at labelling proteins, tests conducted to prove the applicability of CE-LIF to the analysis of samples, and results of a comparison of CE-LIF with some other techniques. Appendices include an instrument operating procedure and a paper that summarizes the experimental details of the protein labelling procedure and a CE-LIF technique for sub-nanomolar assay of proteins.
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ISBN:
Category :
Languages : en
Pages : 43
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Book Description
Capillary zone electrophoresis (CZE), a powerful separation method based on the differential migration of charged species under the influence of an electric field, has been widely used for separations covering from small ions to big biomolecules. Chapter 1 describes the method, then discusses detection of the separated analytes by laser induced fluorescence and by chemical derivatization, and the use of O-phthaldialdehyde (OPA) as a post-column reagent. Chapter 2 describes a post-column reactor which uses two narrow bore capillaries connected coaxially. This reactor differs from other coaxial reactors in terms of capillary dimensions, reagent flow control, ease of construction and most importantly, better limits of detection. The derivatization reagent is electroosmotically driven into the reaction capillary and the reagent flow rate is independently controlled by a high voltage power supply. Amino acids, amines and proteins, derivatized by OPA/2-mercaptoethanol using this post-column reactor coupled with LIF detection, show low attomole mass limits of detection, and for the first time, the authors demonstrate single cell capability with a post-column derivatization scheme. The single cell capability shows that this reactor could find applications in assaying non-fluorescent or electrochemically inactive components in individual biological cells in the future.
Author: Michael D. Harvey
Publisher:
ISBN:
Category : Capillary electrophoresis
Languages : en
Pages : 0
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Book Description
The trend towards high throughput applications and miniaturization necessitates approaches capable of microlitre volume sampling and low protein concentration detection. Furthermore, one of the major trends in high throughput screening is the growing replacement of technologies that depend on radioactivity to generate a signal with those that rely on fluorescence. This trend towards non-radioactive detection in general can be understood by some of the advantages inherent to these methods over radioactive modes. These include a significant reduction in safety concerns leading to a relaxation of strict laboratory procedures, elimination of expensive waste disposal, extended shelf-life of labeled reagents, and the possibility of acquiring multiplexed data through the spectral isolation of different wavelength signals. A variety of capillary electrophoretic (CE) approaches utilizing laser-induced fluorescence (LIF) have thus been developed, providing researchers with valuable tools in protein analysis. Various covalent and non-covalent fluorescent derivatization approaches have been investigated, with emphasis on biochemical and/or clinical applications. The non-covalent dye, NanoOrange, is used as a clinical diagnostic tool for early disease diagnosis, quantitating nanomolar concentrations of human serum albumin in solution, and obtaining fluorescence-based biofluid profiles. An alternate non-covalent labeling approach utilizing the fluorescent probe, Sypro Red, and capillary gel electrophoresis allows for rapid, sensitive analysis of protein sample purity as well as molecular weight determination. These two non-covalent approaches are complemented by the development of a fluorescent Insulin-Like Growth Factor-I (IGF-I) analog for use in bioanalytical applications. Specific derivatization reaction conditions were developed to selectively label the N-terminus of the analog hence preserve biological activity. High-performance liquid chromatography and electrospray mass spectrometry were used to confirm the extent of labeling and modification site. Antibody recognition of this fluorescent analog was evaluated using CE-LIF, illustrating the clinical utility of this diagnostic reagent. In addition to the above CE-LIF approaches, a fourth capillary electrophoretic tool is provided for the clinical chemist. Rapid analysis of biofluids is of significant importance in early disease diagnosis. As such, an extensive CE-based analysis of human seminal plasma is presented. Separation conditions, sample stability, and protein/non-protein zone identification issues are addressed. This study and the CE-LIF methodologies discussed above represent original approaches to nanoscale protein analysis.
Author: Mark A. Strege
Publisher: Springer Science & Business Media
ISBN: 159259798X
Category : Science
Languages : en
Pages : 340
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Book Description
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.
Author: Guido Fridolin Verbeck
Publisher:
ISBN:
Category : Capillary electrophoresis
Languages : en
Pages : 136
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Book Description
Author: H. Shintani
Publisher: Springer Science & Business Media
ISBN: 9400915616
Category : Science
Languages : en
Pages : 750
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Book Description
Over the last decade, high performance Capillary electrophoresis (HPCE) has emerged as a powerful and versatile separation technique that promises to rival high performance liquid chromatography when applied to the separation of both charged and neutral species. The high speed and high separation efficiency which can be attained using any of the various modes of HPCE has resulted in the increased use of the technique in a range of analytical environments. The procedures are, however, still in the early stages of development and several barriers remain to their adoption as the technique of choice for a range of analytical problems. One such barrier is the selection and optimization of the conditions required to achieve reproducible separations of analytes and it is in this area that this new book seeks to give assistance. The book is written by an international team of authors, drawn from both academic and industrial users, and the manufacturers of instruments. At its heart are a number of tables, divided into specific application areas. These give details of published separations of a wide range of archetypal analytes, the successful separation conditions and the matrix in which they were presented. These tables are based on separations reported since 1992 and are fully referenced to the original literature. The tables are supported by discussions of the problems that a particular area presents and the strategies and solutions adopted to overcome them. The general areas covered are biochemistry, pharmaceutical science, bioscience, ion analysis, food analysis and environmental science.
Author: Franklin H. Welder
Publisher:
ISBN:
Category : Capillary electrophoresis
Languages : en
Pages : 290
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Book Description
Author: John R. Petersen
Publisher: Springer Science & Business Media
ISBN: 1592591205
Category : Medical
Languages : en
Pages : 449
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Book Description
Because capillary electrophoresis (CE) is driving the automation of manual and semi-manual techniques, conserves precious samples, and minimizes the use of hazardous organic chemicals, it constitutes a powerful new methodology for the clinical laboratory. In Clinical and Forensic Applications of Capillary Electrophoresis, John R. Petersen and Amin A. Mohammad, along with a panel of leading basic and clinical investigators, review those CE methods that are now replacing many routine serum and blood tests in clinical and forensic laboratories. Major areas reviewed include the coating of columns; the analysis of serum, urine, and CSF proteins and paraproteins; abnormal hemoglobins and hemoglobin Alc; peptides, amino and organic acids; therapeutic drugs; drugs of abuse; viral load; and short tandem repeats (STR). The methods discussed include capillary zone, micellar, electrokinetic, capillary gel, and nonaqueous electrophoresis. The authors show how a variety of detection methods (UV-visible, fluorescence, and MS) can be used in conjunction with CE to detect a broad array of analytes, ranging from serum proteins to PCR products to drugs of forensic importance. Innovative and highly practical, Clinical and Forensic Applications of Capillary Electrophoresis demonstrates the power and versatility of CE-not only to develop new assays, but also to markedly simplify today's clinical and forensic laboratory methodology.
Author: Jianping Ou
Publisher:
ISBN: 9781374748651
Category :
Languages : en
Pages :
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Book Description
Author: Philippe Schmitt-Kopplin
Publisher: Springer Science & Business Media
ISBN: 1588295397
Category : Science
Languages : en
Pages : 808
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Book Description
This book presents a selection of current capillary electrophoresis methods used to separate representative types of molecules and particles and in combination with different detection techniques. It includes practical details which are hard to find elsewhere. The volume is intended for beginners in the field and provides an overview of the technique and a starting point for the exploration of the defined literature on different application topics.
