Electrophoretic gel mobility shift

Figure 4 Effects of p.o.-administered lentinan on the aryl hydrocarbon receptor (AhR) binding activity to xenobiotic responsive element (XRE) and the expression level of CYPIA. Mice were orally administered lentinan and intraperitoneally administered MC as described in Figure 1. Nuclear and microsomal proteins, which were prepared from the livers of the mice, were used for en electrophoretic gel mobility shift assay to determined the AhR binding activity to XRE and for Western blotting analysis to determine the expression level of CYPIA, respectively...

Because conformational changes in RNA or short DNAs typically cause small changes in electrophoretic mobility, analysis of nucleic acid folding requires careful optimization of electrophoresis conditions. By contrast, protein—nucleic acid interactions are typically easier to analyze by native PAGE because the molecular weight and positive charge of the protein produces a relatively large shift in gel mobility. 

In vitro translation is the method of choice for preparing small quantities of protein for analytical purposes and, with the recent availability of commercial reactors and reagents, also for preparative purposes on a modest scale (several mg). Proteins prepared in this way can be analysed by imunoprecipitation or SDS-PAGE they can also be tested for specific biochemical activity, such as enzyme activity or specific DNA binding activity by techniques such as gel electrophoretic mobility shift assays. 

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... 

Assays that exploit the differential electrophoretic mobility of protein-DNA complexes and free DNA are called gel-shift or electrophoretic-mobility-shift assays. In these expert-... 

Four assays are described for archaeal histone binding to DNA. Two are electrophoretic mobility shift assays one using agarose gels to detect compaction of... 

This Iron-Responsive Element (Type II) in the 5 of APP mRNA was fully functional as assessed by multiple separate transfection assays [48]. RNA gel-shift experiments showed that the mutant version of the APP 5 UTR cRNA probe no longer binds to Iron-regulatory Proteins (IRP) (shaded box in Figure 3) [48]. Using RNA electrophoretic mobility shift assays (REMSA) we performed many controls to demonstrate that IRP-1 specifically binds to the stemloop that is predicted to fold from APP 5 untranslated region sequences [48]. Our preliminary data also confirmed that IRP-2 selectively interacted with Ae APP 5 UTR to the same extent as originally observed for IELP-1. 

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