Mobility shift

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... 

Fig. 1 Schematic representation of the experimental setups of the mobility-shift method and the Hummel-Dreyer method (A) the vacancy peak method and the vacancy affinity capillary electrophoresis method (B) the equilibrium-mixture method and the frontal analysis method (C) for drug-protein binding analysis. drug protein gg drug-protein complex Q buffer. (Reprinted with permission from Ref. 38. Copyright 1992 Elsevier Science.)...

In one of the earlier papers, Sun et al. (48) estimated the binding constants of ibuprofen, flurbiprofen, and ketoprofen to HSA and BSA using the mobility-shift mode of ACE. In this case the drugs were actually used as additives to the background buffer to improve the separation of albumin proteins. This is the opposite approach to what is presented in the next... 

The techniques called mobility shift or gel shift assay can be considered a first step in this direction. These are widely used in molecular biology to detect interactions between regulatory proteins for gene expression and specific sequences of polynucleotides (21-23). 

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. 

The examples mentioned up to now utilized strong Ag-Ab interactions using the equilibrium-mixture mode of ACE. But the quantitation of weak antigen-antibody interaction is also possible by ACE, if the mobility-shift approach, sometimes also called dynamic equilibrium affinity electrophoresis, is applied. 

Fig. 6 Evaluation of phosvitin binding to monoclonal anti-phosphoserine antibody by ACE using mobility-shift analysis and UV detection. Peaks M, internal peptide marker mAb, free monoclonal anti-phosphoserine antibody mAb-hpAg complex, monoclonal anti-phosphoserine antibody complexed with homopolyvalent phosvitin antigen. The buffer contained phosvitin within a concentration from 0 to 60 /xM. (Reprinted with permission from Ref. 27. Copyright 1997 Academic Press.)... 

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.)...

The decision whether mobility-shift or equilibrium-mixture analysis... 

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