Mobility shift assay

Cann, J.R., Models of mobility-shift assay of complexes between dimerizing protein and DNA, Electrophoresis, 18, 1092, 1997. 

Cann, J.R., Theoretical studies on the mobility-shift assay of protein-DNA complexes, Electrophoresis, 19, 127, 1998. 

Immediately after a report confirmed that the promoter for SEPSl was stimulated by proinflammatory cytokines, and also showed that this promoter was the target of the critical inflammatory regulator NF-kB. The data included direct binding of this transcription factor to the promoter region using gel mobility shift assays. However, the stimulation of the promoter by cytokines and activation of NF-kB did not result in synergistic production of the mRNA. The role that the cytokines or inflammation plays in regulation of expression of selenoprotein S is not yet clear. 

To investigate the effects of drugs on NFkB activation at the molecular level, the Electric Mobility Shift Assay (EMSA) is a useful read-out system. With this technique the nuclear localization of this transcription factor following activation and subsequent translocation can... 

One of the most impressive examples for the detection of a transcription factor was published in 1996, when a mobility-shift assay in a linear-polyacrylamide-filled capillary using fluorescein-labeled DNA showed 100 times higher sensitivity than the conventional slab-gel technique with 32P. Furthermore, the detection of a transcription factor in a single sea urchin egg was demonstrated (24). 

An ultraviolet (UV) monitor is most commonly used in CE experiment. Such interaction studies using the ACE method can also be hampered by the inadequate sensitivity of UV detection. Fluorescence labeling and laser-induced fluorescence (LIF) detection have been employed to enhance the sensitivity of this method, as shown by the mobility-shift assay of fluorescence-labeled sugar caused by the interaction with the lectin, concanavalin A (74). When fluorescent dyes are employed for labeling, LIF detection provides several hundred times more sensitivity than UV detection. 

Fig. 7 Mobility-shift assay for the determination of dissociation constant of the complex between anti-DNP rat monoclonal IgG21) antibody and charged ligands that contained the A-dinitrophenyl group. Mesityl oxide (MO) served as EOF marker, bovine carbonic anhydrase (CAB) and bovine a-lactalbumin (LA) as internal references. The DNP ligands with a charge of —1 (A) und —9 (B), respectively, were used as additives to the running buffer. (Reprinted with permission from Ref. 30. Copyright 1995 American Chemical Society.)...

Figure 5.4 Detennining the binding affinity of both PNA-1 and PNA-2 with gel mobility shift assay. A fixed concentration of the 32P-labeled 60bp DNA fragment (5 10-9 M) was incubated with increasing concentrations of PNA in TE buffer (lOmM Tris, pH7.5, ImM EDTA) at 37 °C for four hours. The reactants were analyzed by polyacrylamide gel electrophoresis in a 20% gel followed by autoradiography. (A) Binding of PNA-1 to DNA target. (B) Binding of PNA-2 to DNA target.

Determining the binding affinity of both PNA-1 and PNA-2 with gel mobility shift assay 70... 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

The first relevance on pain was activation of PPARa in the spinal cord of rats with peripheral inflammation (Benani et al., 2004). Electrophoretic mobility-shift assay was employed to compare the DNA-binding activity toward a PPRE. The PPARa isoform was observed to be activated in the rat spinal cord after complete Freund s adjuvant injection, which could elicit hyperalgesia. PPARa was provided as a new player in the molecular modeling of pain pathways, although it was discussed that inhibitors of PPARa activation might be relevant antinociceptive drugs. 

The interactions of the G-quadruplex of human telomere DNA with these newly designed molecules have been examined via CD spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative and G-quadruplex or duplex DNA has been investigated by... 

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