Identification of trans-Acting Factors by Electrophoretic Mobility Shift Assay

Cell stimulation with a growth factor or cytokine results in a myriad of intracellular activities, including up- and down-regulation of multiple signal transduction pathways, culminating in observable cellular functions, such as modulation of cell motility, alteration of cell proliferation rate, or induction of apoptosis. Understanding the molecular mechanisms that mediate these cellular functions is of critical importance as aberrations of these pathways lead to human maladies such as autoimmune disease, malignancy, and susceptibility to infection. The most distal components of the signal transduction pathways are the transcription factors. These trans -acting proteins bind to cis-acting DNA elements and, together with the basal transcriptional machinery, control the rate of gene transcription. One of the most useful and common techniques employed in studying transcription factors is the gel mobility shift assay. The overall principle behind this technique involves the use of a radiolabeled piece of DNA mixed with a nuclear protein extract. The protein-DNA complex has a higher molecular weight than the DNA alone resulting in a slower moving or “shifted” band on a nondenaturing polyacrylamide gel ( see Fig. 1 ). These assays have traditionally been used to investigate the binding of novel transcription factors to regions of genomic DNA, typically immediately upstream of the transcription start site of a gene of interest. Using this technique, many ubiquitous and cell type-specific transcription factors as well as their DNA consensus sequences have been described ( 1 – 5 ).

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