| Purification and Quality Control | The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified Gal4-E1A is greater than 90% homogeneous based on SDS-PAGE analysis. | | Unit Definition (Activity) | 1 unit equals 1 nanogram of purified protein. 1 unit is sufficient for a gel mobility shift assay in a 20 µl reaction; 100 units are sufficient for protein-protein interaction assays. | | Applications | GAL4-E1A has been applied in reconstituted in vitro transcription assays and protein-protein interactions assays. | | Formulation and Storage | The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles. | | Background | The GAL4 protein of yeast activates the transcription of several genes involved in galactose metabolism. This event requires that GAL4 bind to upstream activation sites with the consensus sequence 5`-CGGN5(T/A)N5CCG-3` (1 ). A fragment of the GAL4 protein, comprising amino acids 1-147, binds DNA but fails to activate transcription (2). The adenovirus E1A protein stimulates transcription of a wide variety of viral and cellular genes (3). In addition to its trans-activating functions, E1A is also able to modulate progression through the cell cycle (4), to immortalize primary cells in culture (5) and to induce cellular transformation (6). The E1A protein binds to the TATA-binding protein, TBP (7), TAFII55 (8) and TAFII110 (9). Its activating domain has been shown to interact with E2F (10), ATF-2 (11) and YY1 (12). | | References | 1. Kodadek T. (1993) Cell Mol Biol Res. 39, 355-360
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11. Lillie J.W., et al., (1989) Nature 338, 39–44
12. Zhou Q.J., et al., (1995) J. Virol. 69, 7402–7409 |
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