The EMSA (22,23) is used to analyze protein/DNA interactions (Fig. 2). It is based on the observation that when protein is bound to DNA, the mobility of the DNA/protein complex is retarded when compared to the mobility of the free DNA probe. Typically, extracts are prepared from cells transfected with cDNA encoding the DNA-binding protein. This extract is then mixed with a labeled oligonucleotide, and the mixture is run out on a nondenaturing polyacrylamide gel. As a control, extracts are prepared from cells not transfected with the DNA-binding protein, and a sample is prepared that contains no added proteins. Upon autoradiography of the gel, samples containing DNA-binding protein will present bands with significantly reduced mobility than those seen in the control lanes.
To obtain large amounts of TR, cells should be used in which the transfected plasmid containing the TR cDNA is replicated. COS1 cells are a derivative of CVI cells harboring the large T-antigen. Plasmids which contain the simian virus 40 (SV40) origin of replication are able to replicate in these cells. Also HEK 293 cells, which are transformed by adenoviral DNA, allow high expression levels. Another source of TR, TR-variants or TR deletions, is through an in vitro transcription-translation system. Typically, we use the TNT-System from Promega, because in this system, TR cDNAs are efficiently transcribed and translated providing that either a T3, T7, or SP6 RNA polymerase promoter and Kozak sequence are present on the plasmid. It should be noted that for most of the TR binding sites, the heterodimer partner of the TR, RXR, must be present. When extracts prepared from cells are used, the amount of RXR in the extract is usually sufficient to interact with TR and to cause a mobility shift. However, when in vitro translated TR is used, in should be pre-incubated with in vitro translated RXR prior to use in the EMSA.
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