Global analysis electrophoresis

Zuo, X., Speicher, D. W. (2000). A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal Biochem. 284(2), 266-278. 

Proteomics Involves the global analysis of protein expression. In one approach, all the proteins in control cells and treated cells are extracted and subsequently separated using two-dimensional gel electrophoresis. Typically, hundreds or thousands of protein spots are resolved and the steady-state levels of each protein are compared between control and treated cells. In the following example, only a few protein spots are shown for simplicity. Proteins are separated In the first dimension on the basis of charge by Isoelectric focusing (pH 4-10) and then separated by size by SDS polyaciylamide gel electrophoresis. Proteins are detected with a stain such as Coomassle blue and assigned numbers for Identification. 

Lefkovits, I., Kettman, J.R., and Frey, J.R., 2000, Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots. Electrophoresis 21 2688-2693. 

Figure 7.3 Analysis of the 4H four-way RNA junction of the human U1 snRNA by comparative gel electrophoresis (Duckett et al., 1995). The central sequence of the junction is shown. The A G pair at the center was retained in this analysis, although changing it to a Watson—Crick pair did not alter the global shape of the junction. The six long—short species can be considered to be derived from a junction with four arms of 40 bp. The central 20 bp comprises RNA, and the outer arms are DNA. The junction species were electrophoresed in an 8% polyacrylamide gel, in 90 mM Tris—borate (pH 8.3) and 1 mM Mg2+. The mobility pattern of the six species is slow, slow, fast, fast, slow, slow. The simplest interpretation (shown on the right-hand side) is that of a stacked structure based on A on D and B on C coaxial stacking, with the axes nearly perpendicular. The pattern would also be consistent with a rapid exchange between nearly equal populations of parallel and antiparallel forms. However, a recent crystal structure has found a perpendicular stacked structure for this RNA junction (Pomeranz-Krummel et al., 2009).

The capacity of the nanoparticles to adsorb proteins and to activate the complement in vivo after intravenous administration will influence the fate of the carrier and its body distribution. To approach this aspect, in vitro tests have been developed to investigate the profile of the type of serum proteins that adsorbed onto the nanoparticle surface after incubation in serum and to evaluate the capacity of the nanoparticles to induce complement activation. The analysis of the protein adsorbed onto the nanoparticle surface can be performed by 2D-polyacrylamide gel electrophoresis. This technique allows the identification of the proteins that adsorbed onto the nanoparticle surface. To evaluate modifications of the composition of the adsorbed protein with time, a faster method based on capillary electrophoresis can also be used. Finally, the activation of the complement produced by nanoparticles can be evaluated either by a global technique or by a specific method measuring the specific activation... 

The biochemical approach of two-dimensional electrophoresis which has become the classical proteomic approach to whole proteome analysis has the capacity to display a large number of proteins expressed the studied system under given physiological conditions. Construction of global expression maps for defined proteomes is the most widely used application of proteomics and when used in combination with mass spectrometry (MS) techniques can be a powerful approach. There have been a number of studies focused on global neuroproteomics from whole brain analysis to the analysis of synaptic components. Two-dimensional maps have been constructed for whole human (Langen, Bemdt et al. 1999) mouse (Gauss, Kalkum et al. 

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