High-resolution polyacrylamide

Jackson, P. The analysis of fluorophore-labeled glycans by high-resolution polyacrylamide gel electrophoresis. Anal Biochem, 216, 243, 1994. 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

Polyacrylamide shows many advantages over starch gel as a medium for high resolution electrophoresis and because of its synthetic nature its pore size can be more easily controlled. The gel is formed by the polymerization of the two monomers, acrylamide and a cross-linking agent, N, iV-methylene-bis-acrylamide (Figure 3.26). The proportion of the two monomers and not their total concentration is the major factor in determining the pore size, the latter having more effect on the elasticity and... 

The mechanism of separation with linear polymers is as follows. At a certain polymer concentration known as the entanglement threshold, the individual polymer strands begin to interact with each other, leading to a meshlike structure within the capillary. This allows DNA separation to take place. Many of the common polymers are cellulose derivatives, such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and methylcellulose. Other applicable polymers include linear polyacrylamide, polyethylene oxide, agarose, polyvinyl pyrrolidone, and poly-N. Ar-dimethylacrylamide. High-resolution separation up to 12,000 bp has been reported using entangled polymer solutions. 

Isoelectric focusing can be combined with SDS-PAGE to obtain very high resolution separations in a procedure known as two-dimensional gel electrophoresis. The protein sample is first subjected to isoelectric focusing in a narrow strip of gel containing polyampholytes. This gel strip is then placed on top of an SDS-polyacrylamide gel and electrophoresed to produce a two-dimensional pattern of spots in which the proteins have been separated in the horizontal direction on the basis of their pi, and in the vertical direction on the basis of their mass (Fig. 4). The overall result is that proteins are separated both on the basis of their size and their... 

Significant improvements in the technologies of high-resolution two-dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE) and Mass Spectrometry (MS) have marked the start of proteome analysis. Proteomics permits the analysis of thousand of proteins simultaneously, and have the potential to identify markers for early detection, classification and prognosis of diseases, as well as pinpointing targets for improved treatment outcomes [42]. 

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