| Description | The 293 cell nuclear extract was prepared as described (1-3). Although this extract contains all basal transcription factors, as well as most gene-specific activators and cofactors, it is prepared for the purpose of pre-mRNA splicing, especially for cell-specific polyadenylation, DNA replication and pre-mRNA splicing (4,5). 293 cell nuclear extract has been used as the source of individual splicing factors (such as ASF/SF2, see product # P3001 or P3002), and for the cell free system to study the mechanism of pre-mRNA processing (1,3 & 6). | | Source | Extracted from 293 Embryonic Kidney cells. | | Concentration | 6-10 mg protein/ml (in 1x dilution buffer B) | | Unit Definition (Activity) | 1-5 μl is sufficient for a gel mobility shift assay in a 20 μl reaction; 5-10 μl is sufficient for in vitro splicing assay and 20-50 μl is sufficient for a protein-protein interaction assay. Extract activity validated using an in vitro pre-mRNA splicing assay. | | Buffer and Storage | 1x dilution buffer B: 20 mM HEPES-Na (pH 7.9), 20% Glycerol, 42 mM (NH4)2SO4, 0.5 mM DTT and 0.2 mM EDTA.The extract is stored at -80oC. Avoid repeated freezing and thawing cycles. | | Applications | The 293 cell nuclear extract is specifically recommended for 1) in vitro splicing; 2) protein-DNA/RNA and protein-protein interactions; and 3) source of individual splicing factors. Although this extract also contains most transcription factors, it is not recommended for in vitro transcription assays. For Research Use Only. | | References | 1. Noble, J.C.S. et al., (1987) Cell 50, 227-236
2. Krainer, A.R., et al., (1984) Cell 36, 993-1005
3. Ge, H., et al., (1990) Cell 62, 25-34
4. Lewis, E.D., et al., (1985) Nature 317, 172-175
5. Fu, X.Y., et al., (1987) Mol. Cell. Biol. 7, 738-748
6. Fu, X.Y. et al., (1988) EMBO J. 7, 809-817 |
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