Capillary electrophoresis protocol

Wenz, H. M., Dailey, D., and Johnson, M. D. (2001) Development of a high-throughput capillary electrophoresis protocol for DNA fragment analysis, in Capillary Electrophoresis of Nucleic Acids, Vol II. (Mitchelson, K. R., Cheng, J., eds.), Humana, Totowa, NJ, pp. 3-18. 

P. Schmitt Kopplin, Capillary Electrophoresis. Methods and Protocols, Humana Press, Neuherberg (2008). 

Methods development starts with a relatively high number of techniques to characterize and test samples. The number of protocols is often reduced once the critical parameters and the methods that identify them have been defined. The analyst must evaluate the initial techniques with respect to their purposes. If the goal is to generate research data, the practicality of the method and its limitations are not of primary concern if the goal is to use the technique as part of a test procedure, it has to be evaluated in terms of its potential to meet full validation. Critical procedures (e.g., release testing) that cannot be validated will bring a project to an expensive halt. For these reasons, this chapter provides basic principles as well as limitations of capillary electrophoresis (CE) as applied to the analysis of real biopharmaceutical molecules. 

In liquid chromatography, affinity purification protocols (4-8) have been known for a long time. Naturally, electrophoresis can be used just as well to observe molecular or noncovalent interactions of DNA oligomers, provided the complex has distinct electrophoretic properties different from those of the free molecules. Therefore, affinity capillary electrophoresis (ACE) can be a powerful tool for studying DNA-drug or DNA-biopolymer interactions. Several reviews discussing these aspects of ACE have been published in recent years (9-19). The crucial aspects of DNA in this field are covered comprehensively in a recent overview article (20). 

Francois, Y., Zhang, K., Varenne, A., and Gareil, R, New integrated measurement protocol using capillary electrophoresis instrumentation for the determination of viscosity, conductivity and absorbance of ionic liquid-molecular solvent mixtures. Anal. Chim. Acta, 562,164-170, 2006. 

There can be found good reviews on conventional and microchip capillary electrophoresis in forensic/security analysis [4 7] in the literature. The aim of this chapter is to overview the progress which has been made towards the development of portable microfluidic device for on-site and fast detection of nitrated explosives and to describe the major developments in this field (summarized details on analytical methods for microchip determination of nitroaromatic explosives can be found in Table 35.2). The corresponding practical protocol for measurements of explosives on microfluidic device with amperometric detector is described in Procedure 49 (see CD accompanying this book). 

We have carried out an extensive literature search on sample preparation technologies and found many papers on conventional chromatography and capillary electrophoresis methods but few on NLC and NCE. It is important to mention here that sample preparation methodologies used in conventional chromatography and capillary electrophoresis can be used in NLC and NCE. The interested reader can consult our earlier books for details [20,21], However, attempts have been made to describe sample preparation protocols required in NLC and NCE techniques. Some of the important requirements and preparations are discussed below. 

Fig. 11. Principle of synchronized cyclic capillary electrophoresis. Three sample components with different mobility are symbolized. The voltage switching protocol is synchronized to component 2 (reprinted with permission from [73]. Copyright 1994 Hiithig Verlag, Germany)...

Numerous modifications are in place at each lab regarding incubation conditions like substrate concentration, incubation time, cofactor use and concentration, protein concentration etc. These modifications depend on the purpose of the method, e.g. as a high through-put screen in early drug discovery or as a tool to characterize a development compound in vitro for regulatory files. Besides these modifications on the biological part of the assay, specific protocols on pipetting robots and conditions used for evaluation by LC-MS/MS also in combination with the instrumental equipment available are applied which are typically not published. Besides LC-MS/MS also LC-UV (Shearer 2002 Stratford 1999) and capillary electrophoresis (Clohs et al. 2002) are described. 

Orru, S., Pagnozzi, D., and Pucci, R, Interaction proteomics, Biosci. Rep. 25, 45-56, 2005 Zanders, E.D., Ed., Chemical Genomics Reviews and Protocols, Humana Press, Totowa, NJ, 2005 Schou, C. and Heegaard, N.H., Recent applications of affinity interactions in capillary electrophoresis. Electrophoresis 27, 44-59, 2006 Niwayama, S., Proteomics in medicinal chemistry. Mini Rev. Med. Chem. 6, 241-246, 2006 Nedelkov, D. and Nelson, R.W., Eds., New and Emerging Proteomics Techniques, Humana Press, Totowa, NJ, 2006. See also Activity-Based Proteomics. 

As part of the specific transfer, the number of lots, replicates, and injections (in the case of HPLC) should be expressly presented. For dissolution transfers, the number of individual dosage forms that will be tested should be stipulated. It is necessary to spell out such details so that small differences in the everyday analytical philosophies do not perturb the transfer process. In the case of new techniques that may not be common to the receiving laboratory (i.e., capillary electrophoresis or liquid chromatography/mass spectroscopy), specific training may be necessary before the execution of a transfer protocol. 

P. Schmitt-Kopplin, Capillary Electrophoresis Methods and Protocols, Humana, 2007, ISBN 9781588295392 J.P. Landers, Handbook of Capillary Microchip Electrophoresis and Associated Microtechniques, CRC Press, Boca Raton, Florida, 2007, ISBN 9780849333293, C. Henry, Microchip Capillary Electrophoresis, Humana, 2006, ISBN 9781588292933.] The bands are eluted according to their isoelectric points. Isoelectric focusing standards are available which can be used in a preliminary run in order to calibrate the effluent from the column, or alternatively the pH of the effluent is recorded using a glass electrode designed for the purpose. Several efficient commercial equipment are available for separating proteins on a preparative and semi-preparative scale. 

SSCP is based on the conformation-dependent migration of single-stranded DNA (ssDNA) or RNA in native polyacrylamide gel electrophoresis (PAGE) or capillary electrophoresis (CE). The protocol of SSCP analysis used most commonly comprises the following steps (Figure 5.1) ... 

Ruiz-Martinez, M.C., Salas-Solano, O., Carrilho, E., Kotler, L., and Karger, B.L., A sample purification method for ragged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocoL Anal. Chem., 70, 1516, 1998. 

Easley, C. J., Legendre, L. A., Landers, J. R, and Ferrance, J. R, Rapid DNA amphfication in glass microdevices, in Microchip Capillary Electrophoresis Methods and Protocols, C.S. Henry, ed., Humana Press, Totowa, NJ, chap. 15, 2006. 

A. Stoyanov and M. Zhukov, The Proteome Revisited, Isoelectric Focusing J.Chromatography Library Vol 63 2001, Elsevier, ISBN 0444505261, P. Schmitt-Kopphn, Capillary Electrophoresis Methods and Protocols, Humana, 2007, ISBN 9781588295392 J.P. Landers, Handbook of Capillary Microchip Electrophoresis and... 

Tristezza, M., Gerardi, C., Logrieco, A., Grieco, F. (2009). An optimized protocol for the production of interdelta markers in Saccharomyces cerevisiae hy using capillary electrophoresis. Journal of Microbiological Methods, 78, 286-291. 

Capillary Electrophoresis Principles, Instrumentation, and Applications, edited by Kevin D. Altria, 1996 51. Antibody Engineering Protocols, edited by Sudhir Paul, 1995... 

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