Standardization electrophoresis

Ohnesorge, J., Saenger-van de Griend, C., and Waetzig, H. (2005). Quantification in capillary electrophoresis-mass spectrometry long- and short-term variance components and their compensation using internal standards. Electrophoresis 26, 2360—2375. 

Standard electrophoresis on cellulose acetate and citrate agar. 

Linder V, Verpoorte E, de Rooij NF, Sigrist H, Thormann W. Application of surface biopassivated disposable poly(dimethylsiloxane)/glass chips to a heterogeneous competitive human serum immunoglobulin G immunoassay with incorporated internal standard. Electrophoresis 2002 23 740-749. 

Transthyretin amyloidosis (also called familial amyloid polyneuropathy) is an autosomal dominant syndrome characterized by peripheral neuropathy. This disease results from one of five mutations identified thus far in the gene for transthyretin. Transthyretin is also called prealbumin (although it has no structural relationship to albumin) because it migrates ahead of albumin in standard electrophoresis at pH 8.6. Transthyretin is synthesized in the liver and is a normal plasma protein with a concentration of 20-40 mg/dL. It transports thyroxine and retinol binding protein (Chapter 38). The concentration of transthyretin is significantly decreased in malnutrition and plasma levels are diagnostic of disorders of malnutrition (Chapter 17). 

As a standard electrophoresis running buffer for polyacrylamide gels, 1.0-0.5% Tris-borate-EDTA (TBE) is used. To run electrophoresis, use low voltage ranging over 1-8 V/cm to prevent denaturation of small DNA fragments due to high temperature. 

Let us mention that dielectrophoresis has also found wide application in manipulation and sorting of particles and biological cells. Together with standard electrophoresis, it is perhaps the most often used electrokinetic phenomenon with practical applications in mind. Even particle separation can be achieved by using microelectrode arrays [55]. Based on the dielectrophoresis phenomenon, a new technique has recently become available for particle or cell separation, namely the dielectrophoresis/gravitational field-flow fractionation (DEP/G-FFF). In DEP/ G-FFF, the relative positions and velocities of unequal particles or cells are controlled by the dielectric properties of the colloid and the frequency of the applied field. The method has been applied to model polystyrene beads, but, most interestingly, to suspensions of different biological cells [56]. 

Fig. 3. SDS electrophoresis of Lipoproteins I and IL Lipoprotein I (labeled III) was isolated from the hemolymph of a male blue crab and Lipoprotein II (labeled 7) was isolated from the hemolymph of a female crab undergoing vitellogenesis. Label II bands are protein standards. Electrophoresis on 7.5% polyacrylamide gels...

Surface conductance also has pronounced effects on the usual electrophoretic mobUity it can lead to a large drop in the mobUity and therefore to a large error in the reported zeta potential obtained using standard electrophoresis formulae. To determine the true zeta potential in these systems it is necessary to determine the surface conductance. The electroacoustic technique provides the most convenient method for doing this. 

The resulting oligonucleotide is often of surprising purity as judged by analytic HPLC or electrophoresis, and up to 30 mg of a deoxyeicosanucleotide (20-base DNA) can be routinely obtained. Nevertheless small amounts of short sequences, resulting from capping and from base-catalysed hydrolysis, must always be removed by quick gel filtration, repeated ethanol precipitation from water (desalting), reverse-phase HPLC, gel electrophoresis, and other standard methods. 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

The use of standards with samples makes zone electrophoresis particulady usehil as an analytical tool. However, when samples caimot be analyzed on the same gel, differences in the experimental conditions from experiment to experiment make direct comparison more difficult. To make comparisons from experiment to experiment, a relative mobility, is often measured by measuring the distance a component travels down the gel compared to some reference or standard component. 

The used variants of Cg-AR application were adding to the wells of the gel to DNA, directly bringing into the agarose gel and the addition to electrophoretic buffer. The use of the latest way demonstrated its greatest efficiency by saving up to 1.63 times more DNA preparations if compared with the standard method of electrophoresis, while other ways showed 15.45% increase when Cg-AR was introduced into an agarose gel and 1.63%- when added to the DNA preparations. 

The standard Rodbard-Ogston-Morris-Killander [326,327] model of electrophoresis which assumes that u alua = D nlDa is obtained only for special circumstances. See also Locke and Trinh [219] for further discussion of this relationship. With low electric fields the effective mobility equals the volume fraction. However, the dispersion coefficient reduces to the effective diffusion coefficient, as determined by Ryan et al. [337], which reduces to the volume fraction at low gel concentration but is not, in general, equal to the porosity for high gel concentrations. If no electrophoresis occurs, i.e., and Mp equal zero, the results reduce to the analysis of Nozad [264]. If the electrophoretic mobility is assumed to be much larger than the diffusion coefficients, the results reduce to that given by Locke and Carbonell [218]. 

Electrophoretic methods of separation of LD Tsoenzymes have become routine in clinical laboratories. Efforts are now being made to standardize the methodologies used for LD isoenzymes, particularly by Rosalki (38). The preferred methods are based on electrophoresis on a solid medium, so that the several bands may be scanned instrumentally. Differential isoenzyme inhibition with urea or other inhibitors is based on the fact that the heart LD isoenzyme is more resistant to inhibition than other isoenzymes. However, the analyst then has the problem of allocating the observed degree of inhibition between the different isoenzymes of a given sample, a problem that has not been resolved satisfactorily thus far. Hence, differential inhibition is not as reliable for isoenzyme separation as is electrophoresis. 

Rosalki, S. B. Standardization of isoenzyme assays with special reference to lactate dehydrogenase isoenzyme electrophoresis. Clin. Biochem. (1974), 2> 29-40. 

Figure 5 SDS Polyacrylamide Gel Electrophoresis of pectinase from different steps of purification A (1,6) standard protein (2) crude enzyme (3)...

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