Of protein mixtures

Riickert, M., Wohlfarth, M., and Bringmann, G., Characterization of protein mixtures by ion-exchange chromatography coupled on-line to nuclear magnetic resonance spectroscopy, ]. Chromatogr. A, 840, 131, 1999. 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

FIGURE 4.1 2D polyacrylamide gel electrophoresis (2D-PAGE) maps of protein mixtures. (See text for full caption.)... 

Hansen, K.C., Schmitt-Ulms, G., Chalkley, R.J., Hirsch, J., Baldwin, M.A., and Burlingame, A.L. (2003) Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography. Mol. Cell. Proteomics 2, 299-314. 

Since the hydrophobicity of styrene- or alkyl methacrylate-based monolithic matrices is too high to make them useful for hydrophobic interaction chromatography, porous monoliths based on highly hydrophilic copolymers of acrylamide and methylenebisacrylamide were developed [70,135]. The hydrophobicity of the matrix required for the successful separations of proteins is controlled by the addition of butyl methacrylate to the polymerization mixture. The suitability of this rigid hydrophilic monolith for the separation of protein mixtures is demonstrated in Fig. 21, which shows the rapid separation of five proteins in less than 3 min using a steeply decreasing concentration gradient of ammonium sulfate. 

The third test of protein homogeneity, developments from which remain in common use, was that of electrophoresis. Arne Tiselius had been a research assistant in Svedberg s laboratory. From 1925 he pioneered the application of electrophoresis to the analysis and separation of protein mixtures, showing with dialyzed serum differences in mobility of the protein components and the presence of three classes of globulins, a, B, and y. 

Schnabel, R., P. Langer and S. Breitenbach. 1988. Separation of protein mixtures by BIORAN porous glass membranes. J, Membr. Science 36 55-66. 

However, analysis of protein mixtures derived from cells, tissues, and body fluids by 2D PAGE by no means represents a comprehensive picture of the proteins in the mixture. Proteins with extreme isoelectric points, large proteins, small proteins, and hydrophobic proteins are commonly not amenable to 2D PAGE and hence can be easily missed. Furthermore, low abundant proteins are often not detected in 2D gels when proteins of high abundance are present. This limitation is particularly relevant when analyzing serum or other body fluids, where protein amounts vary by ten orders of magnitude (Anderson and Anderson 1998). 

One way of linearizing the problem is to use the method of least squares in an iterative linear differential correction technique (McCalla, 1967). This approach has been used by Taylor et al. (1980) to solve the problem of modeling two-dimensional electrophoresis gel separations of protein mixtures. One may also treat the components—in the present case spectral lines—one at a time, approximating each by a linear least-squares fit. Once fitted, a component may be subtracted from the data, the next component fitted, and so forth. To refine the overall fit, individual components may be added separately back to the data, refitted, and again removed. This approach is the basis of the CLEAN algorithm that is employed to remove antenna-pattern sidelobes in radio-astronomy imagery (Hogbom, 1974) and is also the basis of a method that may be used to deal with other two-dimensional problems (Lutin et al., 1978 Jansson et al, 1983). 

Proteins have different isoelectric points, and in an electrochemical cell they migrate to one of the electrodes (depending on their charge, size, and shape) at different speeds. This difference in behavior is used in electrophoresis for the separation and analysis of protein mixtures. 

The reproducible high-resolution separation of protein mixtures is the main purpose of proteome analysis. O FarelTs classic tube gel technique has limited reproducibility. It is often difficult to compare the protein profiles obtained using O FarelTs method in different laboratories. In some cases, the data obtained even in the same laboratory by different operators are not comparable. 

In Experiment 4, your sample of a-lactalbumin extracted from bovine milk was subjected along with other proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After staining with the dye Coomassie Blue, deeply colored bands appeared on the gel wherever there was a protein. You suspected that some of the blue bands on the gel were due to a-lactalbumin. If molecular weight standards were included on the slab gel, you were able to estimate the molecular weight for a-lactalbumin and other proteins. SDS-PAGE is indeed a very effective analytical tool to achieve fractionation of protein mixtures, to analyze purity, and to estimate molecular weight, but it provides no experimental data to prove the identity... 

Period 1 Fractionation of protein mixture by SDS-PAGE begin passive blot procedure or electroblotting. 

Maizel introduced the use of sodium dodecylsulfate (SDS) for high-resolution electrophoresis of protein mixtures. 

Zone electrophoresis is used mainly as an analytical technique and, to a lesser extent, for small-scale preparative separations. The main applications are in the biochemical and clinical fields, particularly in the study of protein mixtures. Like chromatography, zone electrophoresis is mainly a practical subject, and the most important advances have involved improvements in experimental technique and the introduction and development of a range of suitable supporting media. Much of the earlier work involved the use of filter paper as the supporting medium however, in recent years filter paper has been somewhat superseded by other materials, such as cellulose acetate, starch gel and polyacrylamide gel, which permit sharper separations. 

Marcinkowsky et al. (16) were the first to use dynamic secondary membranes in reverse osmosis for rejection of salts. Giiell et al. (17) later investigated protein transmission and permeate fluxes in microfiltration of protein mixtures using yeast to form a predeposited secondary membrane, and they observed higher flux and protein transmission in the presence of the secondary layer. Kuberkar and Davis (18) also observed higher flux and transmission of BSA in the presence of a cake layer of yeast,... 

The alcohol chloroform precipitate was dislodged, chloroform was removed, the pellet broken up and reextracted with about an equal amount of deionized water by blending the precipitate and the water in a blender and thereafter centrifuging. The reextraction solution was dialyzed and lyophilized with the main extract. If the process proceeds normally, the reextraction of the precipitated hemoglobin usually yields up to 30% of protein mixture present in the original supernatant. An additional reextraction may give an additional... 

One approach to delivering increased performance in a membrane process is to complement one separation mechanism with another. Vapor-arbitrated pervaporation is an example of this strategy. In bioseparations, as will be covered in a later section, a similar integration of several process enhancements in High-Performance Tangential How Filtration is responsible for dramatic improvement in separation efficiency of protein mixtures once considered unachievable by means of conventional ultrafiltration. 

The acid-base properties of proteins are exploited in two methods, electrophoresis and ion exchange chromatography. These are widely used in the analysis and separation of protein mixtures. 

The etched liquid crystal modified capillaries were also tested for the separation of protein mixtures [32], For the two liquid crystal moieties investigated, similar separations of the protein mixtures were obtained. This result is not surprising since previous studies in HPLC have shown that liquid crystals are effective in discriminating between small molecules based on molecular shape [64-66]. The close association of the... 

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