High-resolution polyacrylamide gel electrophoresis

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). 

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]. 

D polyacrylamide gel electrophoresis (2D PAGE) and MS are weU-established and the most commonly employed techniques in proteomics today. 2D PAGE, however, provides limited information of the total amount of proteins. Low-abimdance proteins and small peptides are not detected [1]. Additional methodologies and techniques in sample preparation, selective enrichment, high resolution separation, and detection need to be developed which would allow even higher resolution than 2D PAGE. Acceptable sensitivity to detect the low-abundance proteins is also still an issue. LC can address some of the above-mentioned... 

He, C., Muller, U., Werdan, K. (1992a). Regulation of protein biosynthesis in neonatal rat cardiomyocytes by adrenoceptor-stimulation investigations with high resolution two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 13, 755-756. 

Macfarlane, D.E. (1989) Two dimensional benzyldimethyl-n-hexadecylaimnonium chloride-sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis a high capacity high resolution technique for the purification of proteins from complex mixtures. Anal. Biochem. 176,457-463. 

Figure 11.10. Two-dimensional separation of E. coli protein mixture, using IEF with carrier ampholytes, pH range 3-10, in the first (horizontal) direction, and SDS-PAGE was mn from top to bottom in the second dimension on a 9-14% polyacrylamide gradient running gel cast with 0.1% SDS.12 [Reprinted, with permission, from P. H. O Farrell, The Journal of Biological Chemistry 250 (No. 10 May 25), 1975, 4007 4021. High Resolution Two-Dimensional Electrophoresis of Proteins . Copyright 1975 by the American Society for Biochemistry and Molecular Biology, Inc.]...

Capillary electrophoresis-MS (on-line coupling of CE to ESI-MS, CE-MS) provides high resolution separations of a wide range of proteins (Simo et al., 2004). Two-dimension electrophoresis (2-DE) and MS analysis allow resolution and identification of several thousand proteins. The procedure, which can be automated, involves excision of the protein spots from the 2-DE gel, followed by enzymatic digestion with a protease (e.g. trypsin) and MS analysis (Ashcroft, 2003). 2-DE polyacrylamide gel electrophoresis (2D-PAGE) provides resolution based on both the size and mass differences. 

Melton et al. (1984) observed that RPA allows the detection of as little as 0.1 pg of mRNA, which is at least ten times better than SI analysis. Moreover, RPA are easier and more reliable. Low abundance mRNA are more readily detected than with Northern blotting and quantitation is more accurate. Protected RNA is fractionated by polyacrylamide gel electrophoresis which allows, due to its high resolution, the mapping of the 5 and 3 -ends of the transcripts or the exon/intron boundaries (Calzone et al., 1987 Kekule et al., 1990) and even small differences between probe and target, e.g., after mutations, by adjusting the RNase concentrations (Genovese et al., 1989 Takahashi et al., 1989). 

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