Figure 9.13. Densitometer trace from a stained electrophoresis gel. in .NET

Make Code128 in .NET Figure 9.13. Densitometer trace from a stained electrophoresis gel.
Figure 9.13. Densitometer trace from a stained electrophoresis gel.
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DETECTION OF PROTEINS AND NUCLEIC ACIDS
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The densitometer trace is used to measure peak positions (migration distance) and purity or relative concentrations of components by integration of the areas under the peaks. Modern densitometers perform this operation automatically. For quantitative work, overloading of the gels should be avoided, since this leads to very concentrated sample zones that may inhibit proportional staining and leads to underestimates of sample concentrations. 9.5.2. Detection of Enzymes by Substrate Staining15 Activity stains are of great importance during the isolation, puri cation, and characterization of enzymes, since a particular catalytic reaction is involved and the detection of this activity leads to the unequivocal identi cation of the zone of interest on the electrophoresis gel. Following separation, the gel is removed from the electrophoresis apparatus and is immersed in a minimal volume of a substrate solution. Detection relies on the formation of a colored product by enzyme in the zones containing the enzyme. Examples of activity stains are given in Table 9.2.
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TABLE 9.2. Substrate Stains for Enzyme Detectiona Enzyme Red cell acid phosphatase Phosphoglucomutase Placental alkaline phosphatase b-Glucuronidase Cholinesterase Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Cytochrome oxidase Esterases Lactic and malic dehydrogenases Acid phosphatase Phosphorylase Succinate, b-hydroxybutyric and glutamate dehydrogenase Catalase Caeruloplasmin Leucine aminopeptidase Hemoglobin (as peroxidase)
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Procedures Phenolphthalein diphosphate followed by alkali Reduction of the tetrazolium salt of MTT b-Naphthyl phosphate and diazo salt of fast blue RR 8-Hydroxyquinoline plus blue RR salt 6-Bromo-2-naphthylcarbonaphthoxycholine iodide plus blue B salt Application of agar overlay containing a tetrazolium salt Agar overlay with tetrazolium salt a-Naphthol plus dimethylpapraphenylenediamine a-Naphthylbutyrate plus diazo salt of fast blue RR Reduction of nitro blue tetrazolium salt Sodium a-naphthyl acid phosphate plus diazo salt of 5-chloro-o-toluidine Reduction of silver phosphate to Ag by UV light Reduction of nitro blue tetrazolium salt Inhibition of starch iodide reaction (starch gel) O-Dianisidine Alanyl-b-naphthylamide plus diazotized O-aminoazotoluene Benzidine plus hydrogen peroxide
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See Ref. 15. [Reprinted, with 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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The detection of enzymes by activity staining is important not only for the direct detection of separated enzymes, but also for the detection of noncatalytic proteins after staining with enzyme-labeled antibodies to the protein of interest. The ability to detect a particular catalytic activity greatly reduces background staining, and allows the unequivocal identi cation of a particular analyte species in very complex sample mixtures. 9.5.3. The Southern Blot16 This procedure was introduced by E.M. Southern in 1975, and is used to transfer DNA from agarose gels onto a nitrocellulose or nylon membrane for subsequent detection. Transfer to nitrocellulose is accomplished after the gel has been soaked in a denaturing solution containing $ 1:5 M NaCl and 0.5 M NaOH; this disrupts the base pairing of double-stranded DNA, so that only the single-stranded form is present in the gel. The gel is then neutralized to a pH of $ 8, and placed in a transfer apparatus such as the one shown in Figure 9.14. Once the gel has been placed in the transfer apparatus, buffer in the pan is drawn through the gel by the wicking action of the lter paper. The transfer proceeds over $12 h. The upper lter papers are replaced when they are saturated with buffer. The nitrocellulose membrane is then removed, air-dried and heated to 80  C for 2 h to ensure strong DNA binding. Nylon membranes have largely replaced nitrocellulose for Southern transfers, because nylon has been shown to improve the transfer of small (< 200 base) DNA fragments, and it is more stable to pH extremes. Transfers to nylon follow the same procedure as outlined above, except that NaCl is not needed in the denaturing solution, and no baking step is required to enhance DNA binding to the membrane material. The DNA can be covalently bound to nylon membranes by a brief exposure of the blotted membrane to UV light. These UV-exposed membranes with covalently bound DNA allow multiple detection and rinsing cycles without diffusional zone broadening. 9.5.4. The Northern Blot17 The Northern blot is used to transfer RNA from agarose onto nitrocellulose membranes. Conditions for the transfer are similar to those used in the Southern blot, except for the following. Denaturation prior to transfer is done with methylmercuric
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