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Electrophoresis Experiments (electrophoresis + experiment)

Distribution by Scientific Domains

Chemistry	60%

Selected Abstracts

Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody

ELECTROPHORESIS, Issue 12 2008
Klaus S. Lassen Dr.
Abstract Sulfation is a potentially important post-translational modification of proteins and has been demonstrated in a number of polypeptides, notably in gastrointestinal hormones. In contrast to phosphorylation, however, the investigation of sulfation patterns in tissues and on purified proteins has been complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO3H) antibody) using CE and a panel of sulfated and nonsulfated peptides and proteins. The data show that the anti-Tyr(SO3H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56,65), gastrin-17, and cholecystokinin octapeptide (CCK8) in the 1,3,,M range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a Kd1 of 20.1,,M for the monovalent complex. The same binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody,antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material. Furthermore the specificity of this antibody could be confirmed in one operation using an array of sulfated and nonsulfated compounds. [source]

SDS-CGE of proteins in microchannels made of SU-8 films

ELECTROPHORESIS, Issue 18 2006
Maria Agirregabiria
Abstract This work describes the SDS-CGE of proteins carried out in microchannels made of the negative photoresist EPON SU-8. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology allows the monolithic integration of the electrodes in the device. A high wafer fabrication yield and mass production compatibility guarantees low costs and high reliability. A poly(methyl methacrylate) (PMMA) packaging allows an easy setup and replacement of the device for electrophoresis experiments. In addition, the wire-bonding step is avoided. The electrophoretic mobilities of four proteins have been measured in microchannels filled with polyacrylamide. Different pore sizes have been tested obtaining their Ferguson plots. Finally, a separation of two proteins (20 and 36,kDa) has been carried out confirming that this novel device is suitable for protein separation. A resolution of 2.75 is obtained. This is the first time that this SU-8 microfluidic technology has been validated for SDS-CGE of proteins. This technology offers better separation performance than glass channels, at lower costs and with an easy packaging procedure. [source]

Comparative evaluation of 99mTc-ethylene bis-l-cysteine and 99mTc-ethylene bis-l-,-homocysteine during reversed phase HPLC analysis and electrophoresis at various pH conditions

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 4 2001
K.O. Mang'era
Ethylene bis- L -,-homocysteine (L,L -EH) differs from ethylene bis- L -cysteine (L,L -EC) in having an extra methylene group between each pair of amine and carboxyl groups. The objective of this study was to determine the effect of the extra methylene groups on the characteristics of the complex of these compounds with technetium-99m during analysis by reversed phase HPLC and by electrophoresis at various pH values. Up to pH 5.5, 99mTc- L,L -EH exhibits a substantially longer retention time during reversed phase HPLC than 99mTc- L,L -EC, suggesting a more lipophilic character for 99mTc- L,L -EH under these conditions. On the other hand, 99mTc- L,L -EH clearly possesses a higher negative charge in the pH range 3-6.5 as shown by the markedly greater migration towards the anode in electrophoresis experiments. A rational explanation for these seemingly opposing observations can not yet be offered. Copyright © 2001 John Wiley & Sons, Ltd. [source]

False labelling due to quenching failure of N -hydroxy-succinimide,ester-coupled dyes

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2010
Weiqun Wang
Abstract In comparative fluorescence gel electrophoresis experiments, cross-talk was detected. It was traced back to a failure in the quenching process in typical labelling protocols. Despite a huge excess of potential reaction sites for the N -hydroxy-succinimide,ester-coupled dye, sufficient active dye molecules were available after the quenching step to label protein molecules un-specifically. It could be shown that only a 100-fold increase in the amount of quencher will silence residual dye to such an extent that no artificial signals are detected. [source]

Hierarchical analysis of large-scale two-dimensional gel electrophoresis experiments

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2003
Amit Rubinfeld
Abstract Large-scale two-dimensional gel experiments have the potential to identify proteins that play an important role in elucidating cell mechanisms and in various stages of drug discovery. Such experiments, typically including hundreds or even thousands of related gels, are notoriously difficult to perform, and analysis of the gel images has until recently been virtually impossible. In this paper we describe a scalable computational model that permits the organization and analysis of a large gel collection. The model is implemented in Compugen's Z4000Ô system. Gels are organized in a hierarchical, multidimensional data structure that allow the user to view a large-scale experiment as a tree of numerous simpler experiments, and carry out the analysis one step at a time. Analyzed sets of gels form processing units that can be combined into higher level units in an iterative framework. The different conditions at the core of the experiment design, termed the dimensions of the experiment, are transformed from a multidimensional structure to a single hierarchy. The higher level comparison is performed with the aid of a synthetic "adaptor" gel image, called a Raw Master Gel (RMG). The RMG allows the inclusion of data from an entire set of gels to be presented as a gel image, thereby enabling the iterative process. Our model includes a flexible experimental design approach that allows the researcher to choose the condition to be analyzed a posteriori. It also enables data reuse, the performing of several different analysis designs on the same experimental data. The stability and reproducibility of a protein can be analyzed by tracking it up or down the hierarchical dimensions of the experiment. [source]