Capillary Isoelectric Focusing
Capillary isoelectric focusing (CIEF) is a widespread technique for the analysis of peptides and proteins in biological samples. CIEF is used to separate mixtures of compounds on the basis of differences in their isoelectric point. Aspects of sample prepa
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Capillary Isoelectric Focusing Michał J. Markuszewski, Renata Bujak and Emilia Daghir
Abstract Capillary isoelectric focusing (CIEF) is a widespread technique for the analysis of peptides and proteins in biological samples. CIEF is used to separate mixtures of compounds on the basis of differences in their isoelectric point. Aspects of sample preparation, capillary selection, zone mobilization procedures as well as various detection modes used have been described and discussed. Moreover CIEF, coupled to various types of detection techniques (MALDI or LIF), has increasingly been applied to the analysis of variety different highmolecular compounds. CIEF is considered as a highly specific analytical method which may be routinely used in the separation of rare hemoglobin variants. In addition, the application of CIEF in proteomic field have been discussed on the examples of analyses of glycoproteins and immunoglobins due to the meaning in clinical diagnostic.
7.1 Introduction Capillary isoelectric focusing (CIEF) is an electrophoretic technique used to separate mixtures of compounds on the basis of differences in their isoelectric points (pI). CIEF is generally used to separate amphoteric compounds such as proteins, peptides, and amino acids. The principle of separation in CIEF is shown in Fig. 7.1.
M. J. Markuszewski (&) Faculty of Pharmacy, Medical University of Gdan´sk, Gdan´sk, Poland e-mail: [email protected] R. Bujak E. Daghir Faculty of Pharmacy, Ludwig Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun´, Torun´, Poland
B. Buszewski et al. (eds.), Electromigration Techniques, Springer Series in Chemical Physics 105, DOI: 10.1007/978-3-642-35043-6_7, Springer-Verlag Berlin Heidelberg 2013
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Fig. 7.1 Principle of separation in capillary isoelectric focusing
In CIEF, after filling the capillary with a mixture of solute and ampholytes, the pH gradient is formed. With a basic solution at the cathode and an acidic solution at the anode, upon application of the electric field the charged ampholytes and proteins migrate through the medium until they reach a region where they become uncharged (at pI). There are two modes to perform capillary isoelectric focusing on: • Multi-step CIEF: this mode of CIEF is characterized by presence of focusing and mobilization as two separated steps. Mobilization can be achieved by ion addition or applying a hydraulic force. This mode requires reduced electroosmotic flow (EOF) to a minimum. • Single-step CIEF: in this mode focusing and mobilization steps occur simultaneously. The forces available to transport the zones are the same as for multistep CIEF, but in this mode reduction of electroosmotic flow is not required. Each of these modes need different instrument configurations and different methodologies for optimizing CIEF separation, and they will be discussed in the next parts of this chapter.
7.2 CIEF Methodology 7.2.1 Sample Preparation and Injection Sample preparation for CIEF usually consists of thre
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