Paradigm Gel Shift

Researchers interested in evaluating the interaction of transcription factors with their target sequences commonly employ the electrophoretic mobility shift assay, more commonly known as the "gel-shift." Gel-shift assays can provide crucial information on the binding of transcription factors to their consensus sequences, but because the technique relies on the use of radiolabeled nucleotides, it can be cumbersome and time-consuming. A single gel-shift assay can take days to complete, and in the

Mar 5, 2001
John Piper

Researchers interested in evaluating the interaction of transcription factors with their target sequences commonly employ the electrophoretic mobility shift assay, more commonly known as the "gel-shift." Gel-shift assays can provide crucial information on the binding of transcription factors to their consensus sequences, but because the technique relies on the use of radiolabeled nucleotides, it can be cumbersome and time-consuming. A single gel-shift assay can take days to complete, and in the absence of immunological evidence (i.e., using antibodies for specific transcription factors to "supershift" a complex), results can be hard to interpret. Imagine being able to obtain the same data in a fraction of the time, with both increased specificity and sensitivity. Thanks to the Trans-AM Transcription Factor Assay kit, from Active Motif of Carlsbad, Calif., this dream has now become a reality-if you're investigating the right transcription factors.

Courtesy Active Motif

Active Motif's Trans-AM Assay System
The flagship of the Trans-AM technology is the Trans-AM NF-kB assay. Based on the same principle as the gel-shift, the Trans-AM NF-kB kit relies on the preferential binding of the activated transcription factor to its DNA consensus sequence. One of the keys to the Trans-AM technology is that the target DNA is immobilized in a 96 well plate format, which makes the system ideal for high throughput analyses. After incubating the protein extract with the plate-bound DNA, an antibody is added which recognizes only activated, DNA-bound NF-kB. The entire complex is then detected using an immunological technique that yields a colorimetric signal. Results can be read using a normal microplate reader, so the use of radionucleotides can be totally circumvented.

Hervé Le Calvez, Trans-AM product specialist at Active Motif, explains that the Trans-AM NF-kB assay can be completed in less than four hours, whereas a similar experiment with gel-shift can take up to seven days. In addition to convenience, the Trans-AM technology offers approximately ten-fold more sensitivity than a gel-shift assay, making Trans-AM an attractive alternative to gel-shifts. Currently, the Trans-AM technology is available for the detection of both the p50 and p65 members of the NF-kB family. In the future, expect to see Trans-AM kits for other transcription factors, including AP-1 and CREB.

--John Piper (john.piper@uchsc.edu)

For More Information
Active Motif
(877) 222-9543, www.activemotif.com