The Experts below are selected from a list of 267 Experts worldwide ranked by ideXlab platform
Andreas Chrambach - One of the best experts on this subject based on the ideXlab platform.
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Electroelution without gel sectioning of proteins from sodium dodecyl sulfate polyacrylamide gel electrophoresis fluorescent detection recovery isoelectric focusing and matrix assisted laser desorption ionization time of flight of the electroeluate
Electrophoresis, 2002Co-Authors: Sergey P Radko, Alfred L Yergey, Nancy E Vieira, Sergey F. Zakharov, Huantsung Chen, Ludmila Bezrukov, Andreas ChrambachAbstract:: A method of direct Electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein Electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.
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direct vertical Electroelution of protein from a phastsystem band for mass spectrometric identification at the level of a few picomoles
Proteomics, 2001Co-Authors: Zsuzsanna Buzas, Alfred L Yergey, Huantsung Chang, Nancy E Vieira, Miroslava Stastna, Andreas ChrambachAbstract:An Electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for Electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the Electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus**** (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.
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Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry elements of a new procedure
Electrophoresis, 2001Co-Authors: Alfred L Yergey, Huantsung Chang, Andreas ChrambachAbstract:: Electroelution of protein bands from a gel has advantages over the competitive common technique requiring gel sectioning with respect to yield, speed and the potential for computer-controlled application to multicomponent two-dimensional (2-D) gels. The Electroelution design for the commercial high-performance gel electrophoresis (HPGE) apparatus represented the most advanced technique to date until the recent discontinuation of its production. The present report serves to summarize the necessary design elements for the purpose of renewing and further developing the Electroelution technique. A rudimentary technique is presented by which the electroeluate is collected in a glass tube superimposed on a reversibly stained gel band and connected to an anolyte reservoir. Although the stain used is insufficiently sensitive, the technique allowed for the qualitative verification of its usefulness in the transfer of the electroeluate into mass spectrometry.
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recovery of sodium dodecyl sulfate proteins from gel electrophoretic bands in a single Electroelution step for mass spectrometric analysis
Analytical Biochemistry, 2000Co-Authors: Stanislav Yefimov, Christin Sjomeling, Alfred L Yergey, Tao Li, Andreas ChrambachAbstract:Abstract Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed Electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS–proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive Electroelution steps were required to obviate pH changes in the Electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 μg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single Electroelution step; (ii) using Electroelution from a single gel of 0.3-cm2 cross-sectional area; and (iii) using a protein load of 2 (in one case 4) μg. However, the migration rates of the Cascade blue–SDS–protein–barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.
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Electroelution of nonfluorescent stacked proteins detected by fluorescence optics from gel electrophoretic bands for transfer into mass spectrometry
Electrophoresis, 2000Co-Authors: Erich Gombocz, Stanislav Yefimov, Andreas Chrambach, Alfred L YergeyAbstract:: The extreme accuracy of spectrometrically determined masses of proteins has opened the possibility to identify proteins separated as gel electrophoretic bands in the absence of specific immunologic ways of identification. For the purpose of protein transfer from gel electrophoretic bands to mass spectrometer, Electroelution from the intact gel has advantages, in particular when apparatus with capacity for fluorescent scanning allows one to direct the Electroelution cell over the band under computer control. To avoid fluorescent labeling of the protein which is incompatible with mass spectrometric identification, it is proposed to selectively stack the unlabeled protein and detect it by comigrating tracking dye prior to Electroelution. The feasibility of the approach is exemplified in case of a single protein, but still remains to be demonstrated in conjunction with the selective stacking or unstacking of a single protein from a mixture of several proteins.
Vladimir Besada - One of the best experts on this subject based on the ideXlab platform.
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Sodium dodecyl sulfate free gel electrophoresis/Electroelution sorting for peptide fractionation
IEEE Journal of Solid-state Circuits, 2019Co-Authors: Yassel Ramos, Annia Gonzalez, Yairet Garcia, Aniel Sanchez, Patricia Sosa‐acosta, Yasset Perez-riverol, Lila Castellanos-serra, Luis Javier González, Vladimir BesadaAbstract:Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.
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sodium dodecyl sulfate free gel electrophoresis Electroelution sorting for peptide fractionation
IEEE Journal of Solid-state Circuits, 2019Co-Authors: Yassel Ramos, Annia Gonzalez, Patricia Sosaacosta, Yasset Perezriverol, Yairet Garcia, Lila Castellanosserra, Aniel Sanchez, L Gonzalez, Vladimir BesadaAbstract:Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.
Alfred L Yergey - One of the best experts on this subject based on the ideXlab platform.
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Electroelution without gel sectioning of proteins from sodium dodecyl sulfate polyacrylamide gel electrophoresis fluorescent detection recovery isoelectric focusing and matrix assisted laser desorption ionization time of flight of the electroeluate
Electrophoresis, 2002Co-Authors: Sergey P Radko, Alfred L Yergey, Nancy E Vieira, Sergey F. Zakharov, Huantsung Chen, Ludmila Bezrukov, Andreas ChrambachAbstract:: A method of direct Electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein Electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.
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direct vertical Electroelution of protein from a phastsystem band for mass spectrometric identification at the level of a few picomoles
Proteomics, 2001Co-Authors: Zsuzsanna Buzas, Alfred L Yergey, Huantsung Chang, Nancy E Vieira, Miroslava Stastna, Andreas ChrambachAbstract:An Electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for Electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the Electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus**** (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.
-
Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry elements of a new procedure
Electrophoresis, 2001Co-Authors: Alfred L Yergey, Huantsung Chang, Andreas ChrambachAbstract:: Electroelution of protein bands from a gel has advantages over the competitive common technique requiring gel sectioning with respect to yield, speed and the potential for computer-controlled application to multicomponent two-dimensional (2-D) gels. The Electroelution design for the commercial high-performance gel electrophoresis (HPGE) apparatus represented the most advanced technique to date until the recent discontinuation of its production. The present report serves to summarize the necessary design elements for the purpose of renewing and further developing the Electroelution technique. A rudimentary technique is presented by which the electroeluate is collected in a glass tube superimposed on a reversibly stained gel band and connected to an anolyte reservoir. Although the stain used is insufficiently sensitive, the technique allowed for the qualitative verification of its usefulness in the transfer of the electroeluate into mass spectrometry.
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recovery of sodium dodecyl sulfate proteins from gel electrophoretic bands in a single Electroelution step for mass spectrometric analysis
Analytical Biochemistry, 2000Co-Authors: Stanislav Yefimov, Christin Sjomeling, Alfred L Yergey, Tao Li, Andreas ChrambachAbstract:Abstract Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed Electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS–proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive Electroelution steps were required to obviate pH changes in the Electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 μg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single Electroelution step; (ii) using Electroelution from a single gel of 0.3-cm2 cross-sectional area; and (iii) using a protein load of 2 (in one case 4) μg. However, the migration rates of the Cascade blue–SDS–protein–barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.
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Electroelution of nonfluorescent stacked proteins detected by fluorescence optics from gel electrophoretic bands for transfer into mass spectrometry
Electrophoresis, 2000Co-Authors: Erich Gombocz, Stanislav Yefimov, Andreas Chrambach, Alfred L YergeyAbstract:: The extreme accuracy of spectrometrically determined masses of proteins has opened the possibility to identify proteins separated as gel electrophoretic bands in the absence of specific immunologic ways of identification. For the purpose of protein transfer from gel electrophoretic bands to mass spectrometer, Electroelution from the intact gel has advantages, in particular when apparatus with capacity for fluorescent scanning allows one to direct the Electroelution cell over the band under computer control. To avoid fluorescent labeling of the protein which is incompatible with mass spectrometric identification, it is proposed to selectively stack the unlabeled protein and detect it by comigrating tracking dye prior to Electroelution. The feasibility of the approach is exemplified in case of a single protein, but still remains to be demonstrated in conjunction with the selective stacking or unstacking of a single protein from a mixture of several proteins.
Yassel Ramos - One of the best experts on this subject based on the ideXlab platform.
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Sodium dodecyl sulfate free gel electrophoresis/Electroelution sorting for peptide fractionation
IEEE Journal of Solid-state Circuits, 2019Co-Authors: Yassel Ramos, Annia Gonzalez, Yairet Garcia, Aniel Sanchez, Patricia Sosa‐acosta, Yasset Perez-riverol, Lila Castellanos-serra, Luis Javier González, Vladimir BesadaAbstract:Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.
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sodium dodecyl sulfate free gel electrophoresis Electroelution sorting for peptide fractionation
IEEE Journal of Solid-state Circuits, 2019Co-Authors: Yassel Ramos, Annia Gonzalez, Patricia Sosaacosta, Yasset Perezriverol, Yairet Garcia, Lila Castellanosserra, Aniel Sanchez, L Gonzalez, Vladimir BesadaAbstract:Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.
Goncalo Apolinario De Souza Filho - One of the best experts on this subject based on the ideXlab platform.
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a paper based Electroelution system for protein recovery from stained sodium dodecyl sulfate polyacrylamide gels
Analytical Biochemistry, 2008Co-Authors: Alan T Branco, Beatriz Dos Santos Ferreira, Goncalo Apolinario De Souza FilhoAbstract:Abstract Electroelution is a widely used methodology for protein purification. In this study, a practical and low-cost system for protein Electroelution from stained polyacrylamide gels was developed. For this, a horizontal protein Electroelution cuve was constructed with glass plates, 1.5-ml capacity microcentrifuge tubes, and dialysis membrane. Analyses of the system efficiency showed high protein recovery from nonfixed and fixed sodium dodecyl sulfate (SDS)–polyacrylamide gels.
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A paper-based Electroelution system for protein recovery from stained sodium dodecyl sulfate–polyacrylamide gels
Analytical Biochemistry, 2008Co-Authors: Alan T Branco, Beatriz Dos Santos Ferreira, Goncalo Apolinario De Souza FilhoAbstract:Abstract Electroelution is a widely used methodology for protein purification. In this study, a practical and low-cost system for protein Electroelution from stained polyacrylamide gels was developed. For this, a horizontal protein Electroelution cuve was constructed with glass plates, 1.5-ml capacity microcentrifuge tubes, and dialysis membrane. Analyses of the system efficiency showed high protein recovery from nonfixed and fixed sodium dodecyl sulfate (SDS)–polyacrylamide gels.