Gel-shift analysis

We use the BioRad Miniprotean II gel electrophoresis system for gel shift analysis. The labeling of the DNA fragment that is used as a probe is described for the Southwestern experiment. For binding reactions the probe is diluted with TE to a final concentration of (0.6-1.2) X 10 mol//u.l when 1 /il is added to the 20-fi] reaction, the final probe concentration will be 30-60 pM. This low concentration of probe is necessary to ensure that the protein is in excess over the DNA. This is an important consideration for quantitative gel mobility shift experiments, where approximate dissociation constants (Xd) are determined by using a fixed amount of probe and varying concentrations of protein. Under the condition of protein excess, the is a function of the protein concentration and independent of the DNA concentration and can be estimated from the amount of protein needed to achieve a 50% shift (Ekker et al, 1991). 

Gel-Shift Analysis and Identification of RXREs and RAREs by PCR-Based Selection... 

Fig. I. Gel-shift analysis of a retinoid responsive element of the P-RAR promoter with DR5 configuration that binds RAR and RXR synergistically. The probes were endlabeled with Klenow enzyme and incubated with the bacterially expressed fusion proteins fluRARa [containing the hemaglutinin epitope (14)] and mycRXRa [containing the c-myc 9E10 epitope (12). Coincubation with anti-flu and anti-niyc antibodies respectively results in a disappearance of the heterodimeric complex and a supershift. In contrast, 15C5—a nonrelated monoclonal antibody—does not affect the complex.

Isolate the plasmid DNA by any mimprep procedure and identify the plasmids with inserted oligonucleotides by restnction-digest analysis The insert positive-reporter plasmids can be sequenced according to any of the usual protocols. The selected oligonucleotides can then be functionally tested using either gel-shift analysis or cotransfection experiments. 

More direct evidence for the formation of dimers by the tRNA al. embedded homodimeric maxizyme (tRNAVal Mz Fig.3B)was provided by gel-shift analysis in the absence of the substrate. As controls, we also analyzed tRNAVal transcripts that contained a nonsense sequence between the tRNAVal portion and the terminator sequence, as well as transcripts of the gene for the tRNAVal-embedded parental hammerhead ribozyme (tRNAVal.R32). Shifted bands (dimers) were observed only in the case of the tRNAVal-Mz (Fig.3B). 

Figure 3. The dimeric form of a tRNAV hembedded maxi2yine. (A) A model of the tRNA-embedded dimeric maxizyme (tRNAVafMz). In this model, the internal loop or linker is ignored and is assumed to be an A-form duplex. (B) Detection of the dimeric form of tRNAVal-Mz. Gel-shift analysis revealed that the dimerization occurred only in the case of tRNAV l-Mz, S - P-labeled tRNAV hribozyme (2 nM) was incubated with increasing amounts of the respective non-radiolabeled RNA in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0) at 37 C for 20 minutes. The reaction products were separated on a non-denaturing gel.

S. E., The Gel Shift Assay for the analysis of DNA-protein interactions. In DNA-Protein Interactions Principles and Protocols. Methods in Molecular Biology Vol. 30, Kneale G.G. (Ed.), Humana Press Inc. Totowa, NJ, 1994. 

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... 

DNA Affinity Purification of BUR-Binding Proteins Measuring BUR-Binding Activity by Southwestern and Gel Mobility Shift Analysis... 

Gel Mobility Shift Analysis with Cloned Inserts... 

Fig. 5 Gel mobility shift analysis of total DNA inserts. Insert DNA was purified and labeled as described. Gei mobiiity shift assay was performed as described with purified bacterially produced SATBl. Lanes 1-4 contain 0, 1,2, and 4 nM of SATBl. Gel mobility shift competition results using a. iO-foid excess of unlabeled wild-type (25), (lane 5) and a. 50-fold excess of unlabeled mutated (24)k (lane 6) are shown.

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