Isoelectric Fractionation, Analysis, and Characterization of Ampholytes in Natural pH Gradients. II. Buffering Capacity and Conductance of Isoionic Ampholytes.

Svensson, Harry; Yhland, Margareta; Evans, Warren H.; Sim, G. A.; Theander, Olof; Flood, H.

doi:10.3891/acta.chem.scand.16-0456

Cited by 337 publications

(108 citation statements)

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“…The total volume was 110 ml. 'Ampholine' concentration was 1 or 2°/o, in a sucrose gradient [8]. The pH range varied between 3 and 10 in the different ex periments.…”

Section: Methodsmentioning

confidence: 99%

Heterogeneity of Guinea Pig γ1and γ2 Immunoglobulins

Perini¹,

Binaghi²,

Boussac-Aron³

et al. 1972

Int Arch Allergy Immunol

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Heterogeneity was shown by immunoelectrophoresis in the Fc fragments of normal and anti-DNP guinea pig γ₁and γ₂immunoglobulins, suggesting the existence of subclasses. An attempt was made to isolate the subclasses. Specifically purified anti-DNP antibodies were prepared using 3 different methods, and the γ₁and γ₂classes were separated and analyzed by electrofocusing in a gradient of pH. Resolution of γ₂was not successful: only 1 peak was obtained with an average isoelectric point of 9.4. In the case of γ₁2 populations were separated: γ₁S (slow) average isoelectric point 7.2, and γ₁R (rapid) average isoelectric point 6.4 Immunochemical examination of γ₁S and γ₁R with antisera made in rabbits against each of these fractions indicated that γ₁S consisted of 2 components differing in their Fc fragments, while γ₁R was essentially homogeneous. These results indicate that electrofocusing may be useful to separate the various components of γ₁but the present experiments do not allow a definition of subclasses. The homologous anaphylactic activity (PCA) of γ₁S and γ₁ R was identical on a weight basis.

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“…The total volume was 110 ml. 'Ampholine' concentration was 1 or 2°/o, in a sucrose gradient [8]. The pH range varied between 3 and 10 in the different ex periments.…”

Section: Methodsmentioning

confidence: 99%

Heterogeneity of Guinea Pig γ1and γ2 Immunoglobulins

Perini¹,

Binaghi²,

Boussac-Aron³

et al. 1972

Int Arch Allergy Immunol

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show abstract

“…Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10].…”

Section: -De Historical Evolutionmentioning

confidence: 99%

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

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The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

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“…27 Up to now, several commercial instruments for solution IEF have been developed, including the Rotofor, RF3 (Recycling Free Flow Focusing protein fractionator), the Isoprime, and the IsoelectrIQ2. 9,[27][28][29][30][31][32][33][34][35][36][37][38][39][40]42 In this work, we have developed a method to separate proteins prior to protein digestion and 2D-LC-MS/ MS identification according to their pI, using LIEF in the RF3. Protein pre-fractionation diluted the high abundance proteins by distributing different forms of high abundance proteins into several fractions; the concentration of low abundance proteins could also be increased in the fractionated area, allowing easier detection.…”

Section: Introductionmentioning

confidence: 99%

Prefractionation of Proteome by Liquid Isoelectric Focusing Prior to Two-Dimensional Liquid Chromatography Mass Spectrometric Identification

Zhou

et al. 2005

J. Proteome Res.

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Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC−MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractioantion. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractiontion. LIEF-2D-LC−MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC−MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC−MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges. Keywords: large-scale proteome • fractionation • liquid isoelectric focusing (LIEF) • 2D-LC−MS/MS • low-abundance proteins

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Isoelectric Fractionation, Analysis, and Characterization of Ampholytes in Natural pH Gradients. II. Buffering Capacity and Conductance of Isoionic Ampholytes.

Cited by 337 publications

References 0 publications

Heterogeneity of Guinea Pig γ<sub>1</sub>and γ<sub>2</sub> Immunoglobulins

Heterogeneity of Guinea Pig γ<sub>1</sub>and γ<sub>2</sub> Immunoglobulins

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Prefractionation of Proteome by Liquid Isoelectric Focusing Prior to Two-Dimensional Liquid Chromatography Mass Spectrometric Identification

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