Proteins electrophoretic separation

A PDMS microfluidic system has been reported by Dodge and coworkers [135] that combined on-line protein electrophoretic separation, selection, and digestion of... 

Proteins can ba fractionated by electrophoretic techniques on the basis of one or a combination of their three major properties size, net charge and relative hydrophobicity. Electrophoresis under native conditions is ideal for soluble proteins, where biological properties can often be retained. In contrast, more vigorous and often denaturing conditions must be used for analysis of less soluble proteins. Electrophoretic separations can be carried out using either a continuous or discontinuous (Multiphasic) buffer system. The techniques are referred to as continuous zone electrophoresis (CZE) or discontinuous ("disc") electrophoresis (also known as multiphasic zone electrophoresis, MZE). 

Electrophoresis, a process of separating compounds on the basis of their electric charges, is used to separate and identify mixtures of amino acids and proteins. Electrophoretic separations can be carried out with paper, starch, agar, certain plastics, and cellulose acetate used as solid supports. In paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels (Figure 18.4). Next, a sample of amino acids is applied as a colorless spot on the paper strip. (The amino acid... 

Charged macromolecules, such as proteins or polymers, are often separated elec-trophoretically. The rate of migration through an electric field increases with net charge and field strength. Molecular size of analytes and viscosity of separation media both have inverse relationships with rate of migration. These variables must all be taken into account in order to optimize the conditions for an efficient electrophoretic separation. 

Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 fim nitrocellulose membranes. After transfer of proteins, membranes were... 

P. Juo and G. Stotzky, Electrophoretic. separation of proteins from roots and root exudates, Canadian Journal of Botany 48 1 7i (1970). 

Chemical surface modifications The first surface modification for the purpose of eliminating EOF and protein adsorption was recommended by Hjerten.28 The attachment of vinyl silanes allowed the polymerization of a variety of molecules to the surface. Most of the chemical modifications used for preparing capillaries for electrophoresis originated from the experience acquired over the years preparing GC and LC stationary phases. Chemical modification should conform to certain requirements, including the prevention of adsorption, the provision of stable and constant EOF over a wide pH range, chemical stability, ease of preparation, and reproduciblity of preparation. The effects of silanization of the inner surface of capillaries on electrophoretic separations have been extensively studied.26-29... 

McCormick, R. M., Capillary zone electrophoretic separation of peptides and proteins using low pH buffers in modified silica capillaries, Anal. Chem., 60, 2322, 1988. 

Towns, J. K. and Regnier, F. E., Capillary electrophoretic separations of proteins using nonionic surfactant coatings, Anal. Chem., 63, 1126, 1991. 

Emmer, A., Jansson, M., and Roeraade, J., Improved capillary zone electrophoretic separation of basic proteins, using a fluorosurfactant buffer additive,... 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Shadpour, H., Soper, S.A. (2006). Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips. Anal. Chem. 78, 3519-3527. 

Hj erten (1983) reported the use of crosslinked polyacrylamide gels for the capillary electrophoretic separation of proteins. However, crosslinked polymers are quite rigid, and the capillary has a short lifetime. 

Norbeck J et al. Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension reproducibility and evidence for isoelectric focusing of alkaline (pi >7) proteins. Yeast 1997 13 1519-1534. 

Obviously, the main purpose for the introduction of CL detection coupled to CE separations is inherent to the development and improvement of sensitive and uncomplicated devices to achieve a decrease of the band broadening caused by turbulence at the column end, together with the attractive separation efficiency of CE setups. With this purpose in mind, Zhao et al. [83] designed a postcolumn reactor for CL detection in the capillary electrophoretic separation of isoluminol thiocarbamyl derivatives of amino acids, because, like other isothiocyanates, isoluminol isothiocyanate has potential applications in the protein-sequencing area. 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

Direct staining of proteins (e. g., after electrophoretic separation in polyacrylamide gels) can be achieved by treatment with dyes like Coomassie Brilliant Blue R-250 [146] (Fig. 7), which binds positively charged proteins in an acidic fixation buffer, allowing detection down to 0.1 pg of protein. 

Fig. 7. Coomassie Brilliant Blue R-250 (I) is used to stain proteins, e.g., after gel-electrophoretic separation, its derivative G-250 (II) is applied in the Bradford assay for protein quantification... 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

Figure 11.15 Immunoelectrophoresis of human serum proteins. The proteins are separated electrophoretically from wells cut in a suitable gel. After electrophoresis, a trough is cut in the gel parallel to the direction of migration and filled with an antiserum. The components are allowed to diffuse for 24-48 hours for precipitation lines to develop. Human serum contains many proteins, among which the immunoglobulins can be identified.

IEF is an electrophoretic method in which proteins are separated on the basis of their pis.1 214-6 10 64-69 p makes use of the property of proteins that their net charges are determined by the pH of their local environments. 

Isoelectric Focusing This is an electrophoretic method in which the proteins are separated based on their charge characteristics. This is accomplished by the proteins moving through a medium with a pH gradient. 

SDS-PAGE has traditionally been used as the primary method for size-based protein separations. SDS binds to polypeptide chains so that similar charge densities and constant mass-to-charge ratios of proteins are obtained. Then, electrophoretic separation of SDS-protein complexes based on size is achieved in a sieving medium. Detection of the separated... 

In CZE, proteins are separated from each other based on the differences in their electrophoretic mobilities. The electrophoretic mobility is a function of the molecular charge and hydrodynamic size of a protein. In a given environment, the electrophoretic mobility is an intrinsic property of the protein, similar to the isoelectric point. Therefore, the mobility can be used to distinguish proteins from each other. This is the basis for using CZE as a simple identity test for final product and package labeling. As an example, the CZE profiles of six... 

Shadpour, H. Soper, S. A. Two-Dimensional Electrophoretic Separation of Proteins using Poly(methyl methacrylate) Microchips. Anal. Chan. 20 06, 78, 3519-35 27. 

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