Band-shift assay,

The electrophoretic mobility shift assay (EMSA), also called the gel-shift or band-shift assay, is more useful than the footprinting assay for quantitative analysis of DNA-binding proteins. In general, the electrophoretic mobility of a DNA fragment is reduced when it is complexed to protein, causing a shift in the location of the fragment band. This assay can be used to detect a transcription factor in protein... 

Identify the probes that are retarded by the BUR-binding protein (Fig. 5), isolate them, and amplify once again (Fig. 6). To obtain sufficient amounts for isolation, repeat the band shift assay in a scaled-up reaction (for example, 10 times) and separate the entire sample in all 10 lanes of the miniband shift gel. 

Molekularsiebchromatographie, Gelpermeations-Chromatographie gel point Gelpunkt gel retention analysis/ band shift assay Gehetentionsanalyse... 

Figure 2.10. Electorphoretic mobility shift assay (EMSA). C/EBPP is a basic leucine transcription factor. An DNA oligomer consisting of the C/EBP binding site was labeled with 32P and incubated with a nuclear extract (lanes 1 and 2). The same oligomer and nuclear extract were incubated in the presence of a C/EBPP antibody (lanes 4 and 5). In lanes 1 and 2, the unbound free probe can be seen at the bottom of the autoradiogram, while probe protein complexes can be visualized in the upper section. In lanes 4 and 5 the presence of the antibody resulted in the supershift of the protein-probe complex the ternary complex consisting of the probe, C/EBPP protein, and C/EBPP antibody are now visualized at the top of the autoradiogram. The bands that were supershifted with the antibody represent C/EBPp.

Treatment of this polymer with TMSI under the same conditions employed for the reaction with S-b-tBM resulted in a quantitative production of MM-b-MA. The t-butyl signal in the NMR spectrum is now gone (Figure 3b), and the carbonyl band in the IR spectrum is further broadened and shifted to 1717 cm (Figure 4b). Titration for MA resulted in 0.583 meq COOH/g, in accord with the value of 0.56 meq/g calculated based on the amount of tBM present in the NMR spectrum. Conversion to the potassium methacrylate copolymer was straightforward. IR analysis of the product shows the carboxylate band at 1552 cm-1, and the ester band at 1729 cm-1 (Figure 4c). Assay for potassium (ICP) confirmed that the neutralization was quantitative. 

Like the nicotinamide coenzymes (Fig. 13-15), the flavin nucleotides undergo a shift in a major absorption band on reduction. Flavoproteins that are fully reduced (two electrons accepted) generally have an absorption maximum near 360 nm. When partially reduced (one electron), they acquire another absorption maximum at about 450 nm when fully oxidized, the flavin has maxima at 370 and 440 nm. The intermediate radical form, reduced by one electron, has absorption maxima at 380, 480, 580, and 625 nm. These changes can be used to assay reactions involving a flavoprotein. 

Dye-Binding (Bradford) Assay. The binding of proteins to Coomassie Brilliant Blue 250 causes a shift in the absorbance maximum of the dye from 465 nm to an intense band at 595 nm. Determination of the increase in absorbance at 595 nm as a function of protein added provides a sensitive assay... 

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