Sodium dodecyl sulfate-polyacrylamide applications

Castellanos-Serra, L.R., Femandez-Patron, C., Hardy, E., Santana, H., Huerta, V. High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins. J Protein Chem 16 (1997) 415—419. 

Some areas of investigation are restricted by the requirement for purified proteins because these may be limited in quantity of source material and their purification often involves tedious and inefficient procedures. Elder et al. have reported a unique approach that bypasses some of these restrictions in their studies of viral and plasma membrane proteins. After separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they remove the gel slice containing the protein band of interest and radioiodinate it by chloramine-T while still in the gel. This application should prove to be useful in other systems. 

It is a detergent and wetting agent effective in both alkaline and acidic conditions. In recent years it has found application in analytical electrophoretic techniques SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis is one of the more widely used techniques for the analysis of proteins and sodium lauryl sulfate has been used to enhance the selectivity of micellar electrokinetic chromatography (MEKC). ... 

Electrophoretic assays may be used to characterize the purity and homogeneity of biopharmaceuticals, based mainly on an ability to differentiate chemical and molecular changes in the compound as a result of oxidation, denaturation, aggregation, and deamidation [11]. Well-established techniques include isoelectric focusing (lEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whilst high-performance capillary electrophoresis (HPCE), utilizing a variety of detection systems, may also be used for routine applications. 

Most of the applications of HPLC for protein analysis deal with the storage proteins in cereals (wheat, corn, rice, oat, barley) and beans (pea, soybeans). HPLC has proved useful for cultivar identihcation, protein separation, and characterization to detect adulterations (illegal addition of common wheat flour to durum wheat flour) [107]. Recently Losso et al. [146] have reported a rapid method for rice prolamin separation by perfusion chromatography on a RP POROS RH/2 column (UV detection at 230nm), sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and molecular size determination by MALDl-MS. DuPont et al. [147] used a combination of RP-HPLC and SDS-PAGE to determine the composition of wheat flour proteins previously fractionated by sequential extraction. 

Figure 12.12b illustrates the application of gel electrophoresis to protein characterization. In this illustration a cross-linked polyacrylamide gel is the site of the electrophoretic migration of proteins that have been treated with sodium dodecyl sulfate. The surfactant dissociates the protein molecules into their constituent polypeptide chains. The results shown in Figure 12.12b were determined with well-characterized polypeptide standards and serve as a calibration curve in terms of which the mobility of an unknown may be interpreted to yield the molecular weight of the protein. As with any experiment that relies on prior calibration, the successful application of this method requires that the unknown and the standard be treated in the same way. This includes such considerations as the degree of cross-linking in the gel, the pH of the medium, and the sodium dodecyl sulfate concentration. The last two factors affect the charge of the protein molecules by dissociation and adsorption, respectively. Example 12.5 considers a similar application of electrophoresis. 

Capillary gel electrophoresis [55,56] (CGE) is very similar to CZE. The main difference is that in CGE the column is packed with a gel, which affects the motion of the analytes. Accordingly, separation will be determined not only by the electrophoretic force acting on the ions but also by the size of analyte molecules. The effect of the gel present inside the column has a similar effect to size exclusion chromatography (see earlier). Atypical application is the separation of proteins in a capillary which is filled with polyacrylamide gel and sodium dodecyl sulfate (SDS). The presence of SDS aids the electrophoretic mobility of proteins, as it coats their surface proportional to their size. Consequently, the molecular structure will have little influence on mobility, so macromolecules will migrate according to their molecular mass. This technique is very similar to SDS-PAGE. 

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