PRINCIPLES OF ELECTROPHORESIS in .NET

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Figure 9.11. Comparison of the band separation of linear, wedge, and ionic strength gradient gels. The T reaction of a sequencing experiment carried out on M13 mp8 DNA was run on three different 40 cm 6% polyacrylamide gels, using (a) a standard linear gel, (b) a 0.35 1.05-mm wedge gel, and (c) a 0.05 0.50 M Tris buffer gradient. [Reprinted, with permission, from A. T. Bankier and B. G. Barrell, in Nucleic Acids Sequencing: A Practical Approach , C. J. Howe and E. S. Ward, Eds., Oxford University Press, New York, 1989. # IRL Press at Oxford University Press 1989.]
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mixtures (the T reaction) using a 6% polyacrylamide gel in the linear, wedge and ionic strength gradient con gurations is shown in Figure 9.11. 9.5. DETECTION OF PROTEINS AND NUCLEIC ACIDS AFTER ELECTROPHORETIC SEPARATION Following an electrophoretic run, the band from the tracking dye is often the only visible band. The detection of separated proteins and nucleic acids requires subsequent treatment of the separation pattern for visualization. This treatment may be performed directly on the gel, or may require a blotting step in which the entire separation pattern is transferred onto a thin membrane material. The choice of detection method depends on the concentrations of analytes in the separated zones and whether recovery of the puri ed sample is required.
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9.5.1. Stains and Dyes13 The most common visualization method involves staining an entire gel with a species that interacts in a nonselective way with either proteins or nucleic acids. Protein gels are removed from the electrophoresis apparatus and xed by immersion in 10% trichloroacetic acid, to precipitate proteins and prevent diffusional zone broadening and analyte loss into staining solutions. The gel is then rinsed, and immersed in a solution (0.1 0.2% w/v) of the stain, resulting in stain absorption throughout the gel. A destaining rinse step then leaves stain only where interactions with protein prevent its removal. Similar procedures are used for the detection of nucleic acids. Table 9.1 lists stains for proteins, glycoproteins, and nucleic acids. A silver stain has been introduced that is 100 times more sensitive for proteins than Coomassie Blue, and 4 times more sensitive for nucleic acids than ethidium bromide.14 This stain is based on the reduction of Ag by thiol, tyrosine and amine functional groups in proteins and by the purine bases in DNA. The solid silver formed by reduction precipitates in the gel forming a permanent stain in the protein nucleic acid zones, and is not reduced by buffer or gel materials, so that
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TABLE 9.1. Electrophoresis Stainsa Stain Amido Black 10B Coomassie Brilliant Blue R-250 Coomassie Brilliant Blue G-250 Alcian Blue Uniblue A Methylene Blue Methyl Green Fast Green FCF Basic Fuschin Pyronin Y Bromophenol Blue Bromocresol Green Crocein Scarlet Xylene Cyanole FF Toluidine Blue O Ethidium Bromide Stains All
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Absorption Max. (nm) Use 620 590 General protein stain General protein stain, 10 times more sensitive than Amido Black General protein stain Glycoprotein Protein stain RNA, RNase Native DNA, neutral or acidic tracking dyes Protein stain Glycoprotein, nucleic acids, sialic acid-rich glycoproteins RNA, acidic tracking dye Neutral and alkaline tracking dye Tracking dye for DNA agarose electrophoresis Immunoelectrophoresis Tracking dye for DNA sequencing RNA, RNase, mucopolysaccharides Fluorometric detection of DNA General protein stain
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See Ref. 13. [Reprinted, by permission, from P. G. Righetti, Isoelectric Focussing: Theory, Methodology and Applications , Elsevier, New York, 1983. # 1983, Elsevier Science Publishers B.V.]
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PRINCIPLES OF ELECTROPHORESIS
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Figure 9.12. Comparison of identical polyacrylamide gels stained with Coomassie Blue (lanes 1 3) and Silver Stain (lanes 4 6). The initial sample concentrations are identical for lanes 1 and 4, 2 and 5, and 3 and 6. [Reprinted, with permission, from B. S. Dunbar, TwoDimensional Electrophoresis and Immunological Techniques , Plenum Press, New York, 1987. # 1987 Plenum Press.]
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background staining is minimal. Figure 9.12 shows a comparison of identical gels stained by coomassie blue and a silver stain. After staining, gels may be photographed under UV or visible light, to yield qualitative information about the number and positions of the bands. Alternatively, quantitative information can be obtained from a scanning densitometer (Fig. 9.13) that measures light transmitted through the gel as a function of position in the gel.
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