| Purification and Quality Control | The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified GAL4-AH 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-AH has been applied in 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 | Transcriptional activity is greatly stimulated by promoter-specific activator proteins (1, 2). These are modular proteins, consisting of a DNA-binding domain and a regulatory (activator) domain (3, 4). 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` (5 ). A fragment of the GAL4 protein, comprising amino acids 1-147, binds DNA but fails to activate transcription (6). Linking of an acidic synthetic peptide, forming an α-helix (AH), to this GAL4 DNA-binding domain, results in a protein with an amphipathic structure (7). This fusion protein is able to activate transcription of a gene, bearing the GAL4 binding sites in an in vitro transcription system by targeting TFIIB in the pre-initiation complex (7-9). | | References | 1. Lewin, B. (1990) Cell 61, 1161-1164
2. Ptashne, M., et al., (1990) Nature 346, 329-331
3. Ma, J., et al., (1987) Cell 48, 847-853
4. Ma, J., et al., (1987) Cell 51, 113-119
5. Kodadek T. (1993) Cell Mol Biol Res. 39, 355-360
6. Keegan, L., et al., (1986) Science 231, 699-704
7. Giniger, E., et al., (1987) Nature 330, 670-672
8. Lin, Y-S., et al., (1988) Cell 54, 659-664
9. Lin, Y-S., et al., (1991) Cell 64, 971-981 |
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