D Gel Electrophoresis

When fresh or frozen tissue is used for proteomic analyses, the results cannot be related directly to the clinical course of diseases in a timely manner. Instead, researchers frequently reduce the number of interesting proteins to a manageable number and then attempt to use immunohistochemistry to understand the implications of proteomic changes in archival formalin-fixed, paraffin-embedded (FFPE) tissue for which the clinical course has been established.3 Unfortunately, immunohistochemistry is a semiquantitative pro-teomic method, and the choice of interesting proteins must occur without advance knowledge of the clinical course of the disease or the response to therapy. If routinely fixed and embedded archival tissues could be used for standard proteomic methods such as 2-D gel electrophoresis and mass spectrometry (MS), these powerful techniques could be used to both qualitatively and quantitatively analyze large numbers of tissues for which the clinical course has been established. However, analysis of archival FFPE tissues by... 

High-throughput proteomic methods hold great promise for the discovery of novel protein biomarkers that can be translated into practical interventions for the diagnosis, treatment, and prevention of disease. These approaches may also facilitate the development of therapeutic agents that are targeted to specific molecular alterations in diseases such as cancer. In many cases, malignant cells yield unique protein profiles when total protein extracts from such cells are analyzed by 2-D gel electrophoresis or mass spectrometry (MS) methods. Such proteomic studies have the potential to provide an important complement to the analysis of DNA and mRNA extracts from these tissues.1... 

Packed capillaries with a larger inner diameter may be useful in preparative separations. These will find an application in proteome research as a part of multidimensional separation systems that will replace 2-D gel electrophoresis. The preparative CEC will require solving of the problems related to heat dissipation since the radial temperature gradient negatively affects the separations, and sample injection. The fabrication of sintered frits in larger bore capillaries is also very difficult. However, in situ polymerized monolithic frits can be fabricated in capillaries of virtually any diameter [190]. 

Huber s group recently prepared poly(styrene-co-divinylbenzene) monolithic columns in the capillary format using tetrahydrofuran/decanol mixtures as poro-gen. These columns were tested for the HPLC separation of protein digests followed by ESI MS detection enabling protein identification [129]. This technique represents an important contribution to the currently emerging techniques for studying of proteomes as it is more convenient and accurate to use than the classical 2-D gel electrophoresis. 

Joo WA, Lee DY, Kim CW. Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D gel electrophoresis. Biosci Biotechnol Biochem 2003 67(7) 1574-1577. 

Albumin can be measured quantitatively by the bromcresol green method in most species (Evans and Duncan 2003). Other proteins (described below) are measured by immunometric methods. Newer methods based on proteomics technology (concentration of proteins by acetone precipitation or ultracentrifugation, separation by 2-d gel electrophoresis or chromatographic techniques with subsequent identification and quantitation by mass spectrometry) have been used experimentally (Bandara and Kennedy 2002 Chapman 2002 Thongboonkerd et al. 2002a, b). 

Figure 15.1 General scheme showing the steps to be followed for the preparation of cytosolic, mitochondrial, and membrane brain proteins and for the enrichment of the cytosolic proteins prior to the 2-D gel electrophoresis analysis.

M. Hamdan and P. G. Righetti, Assessment of protein expression by means of 2-D gel electrophoresis with and without mass spectrometry, Mass Spectrom. Rev. 22 (2003), 272-284. 

The study of protein structure, function, quantity, and interactions during maturation and progression of disease is referred to as proteomics. Analytical approaches that use a combination of two-dimensional (2-D) gel electrophoresis for protein separation and MS analysis for protein identification followed by database searches is a widely practiced proteomics strategy.The tryptic peptides extracted from gels are analyzed by MALDI-TOF MS and microcolunm or capillary LC tandem mass spectrometry (MS/MS) techniques. Typically, the MALDI-TOF MS techniques are used to quickly identify peptide fragments and confirm the presence of known proteins. Nano-scale capillary LC/MS/MS techniques (using 50-100 pm diameter columns, operating at flow rates of 20-500 nL/min) are... 

Gersten D. Gel Electrophoresis of proteins Essential Techniques Series. New York John Whey 8 Sons, 1996. Harris DC. Principles of capillary electrophoresis. In Harris DC. Quantitative chemical analysis. New York ... 

Jones P, Rickwood D. Gel electrophoresis Nucleic acids. Essential Techniques Series. New York John Wdey Sons, 1995. 

Gersten D. Gel electrophoresis of proteins Essential techniques, New York John Wdey St Sons, 1996. 

In order to establish a molecular data base for BR and begin to address questions (a) and (b) above, we have examined whether BR affects the transcription of auxin-induced genes in elongating soybean epicotyl sections using available auxin-induced soybean sequences as probes. More generally, we have shown changes in protein synthesis caused by BR in soybean epicotyls and hypocotyls by in vitro translation of mRNA (+ or - BR) followed by 2-D gel electrophoresis. 

Figure 3. 2-D Gel Electrophoresis of in vitro Translated Soybean Hypocotyl mRNA. Numbers to the left indicate migration of molecular weight markers (kilodaltons). The separating gel was 12% polyacrylamide. Numbered arrows indicate polypeptides that are up-regulated by BR while lettered arrows show polypeptides that are down-regulated in response to BR. Other experimental details are described in the text. A = hypocotyl sections auxin-depleted for 2 hours followed by buffer treatment for 2 hours A = as in A with 340 nM BR replacing buffer treatment.

Figure 4. 2-D Gel Electrophoresis of in vitro Translated Soybean Epicotyl mRNA. Experimental conditions are as described in Figure 3. B = epicotyl sections auxin depleted for 2 hours followed by incubation with 5 x 10"5 M 2,4-D for 2 hours B = epicotyl sections auxin depleted as in B followed by incubation with 3.4 x 10 7 M BR for 30 minutes with a further treatment of both 3.4 x 10-7 M BR and 5 x 10 5 M 2,4-D for 2 hours.

Overproduction of EPSPS has been observed in several plant cell cultures tolerant to glyphosate (12, 13, 29). In the case of glyphosate-tolerant Corydalis cultures, Smart et al. demonstrated by 2 D-gel electrophoresis, the overproduction of other proteins besides EPSPS. Since the levels of activity of several shikimate pathway enzymes were unaltered in the tolerant cell line compared to the parent cell line, it was concluded that these amplified proteins may not be involved in aromatic amino acid biosynthesis. It is possible that the other proteins may not have a role in the tolerance mechanism. Alterations in protein profiles between glyphosate-sensitive and tolerant petunia cell lines have also been observed. With the glyphosate tolerant carrot cell line, in addition to overproduction of EPSPS, the levels of aromatic amino acids were found to be enhanced (29). Based on the results with plant cell cultures, it was therefore not clear if overproduction of EPSPS was sufficient to obtain glyphosate tolerance in plants. 

D gel electrophoresis is currently a major tool in the field of proleomlcs which, by analogy with genomics, is allempling lo map all the proteins m a living organism. See also Chapter 3. 

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