The first stage of this assay establishes the binding activities of TBP required to fully shift 0.2pmol of the TATA-containing oligonucleotide in 25 l of binding buffer containing MgCl2. Next, binding reactions are performed in the absence of MgCl2. In the absence of Mg ions, the binding of TBP to DNA is weakened enough that not all of the DNA shifts, even though the previous experiment extablished that a stoichiometric amount of active TBP is present. Although this unit is desgnated as a gel shift unit, in this case it really is a TBP-binding unit as detected by gel shift. In vitro transcription reactions were performed as described in reference 9.
Bars indicate the high and low values of replicate samples.
Gel shift assays were performed as described in this article. Promega also routinely tests transcription factor preparations for DNase and RNase activities. Any DNase activity in the transcription factor preparations could certainly confound results obtained from footprinting and gel mobility shift assays, and Rnase contamination could adversely affect studies in which the factor is added to reconstituted systems for transcriptional assays. Specific antibodies against general transcription factors can be used for several purposes. For example, an antibody can be used to follow the purification of a factor from nuclear extracts or recombinant sources, to purify with antibody-coupled resins, to immunoprecipitate and to verify the presence of the factor in gel shifted complexes.