Description:The HeLa cell nuclear extract, catalog number P0001, is specifically recommended for: 1) in vitro transcription, 2) protein-DNA/RNA and protein-protein interactions, and 3) as a source of individual transcription factors. Although this extract also contains most pre-mRNA processing factors, it is, however, not recommended for in vitro pre-mRNA processing.
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| Description | The HeLa cell nuclear extract was prepared as described by Dignam et al. (1) and Manley et al. (2). This extract contains all basal transcription factors, including TFIIA, -IIB, -IID, -IIE, -IIF, -IIH and RNA polymerase II, as well as most gene-specific activators and cofactors, such as Sp1, Oct-1, NF-kB, USF, ATF, PC4, p300 etc. (3-7). Therefore, it has been used as the source of transcription factors for the cell-free transcription system to study specific transcription by RNA polymerase II as well as RNA polymerase III (8) and for purifying transcription factors (9, 10). | | Source | Extracted from Cervical Tumor cells | | Concentration | 8 mg protein/ml (in 1x dilution buffer A) | | 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 transcription assay and 20-50 μl is sufficient for a protein-protein interaction assay. Extract activity has been validated using an in vitro transcription assay. | | Buffer and Storage | 1X dilution buffer A: 20 mM Tris-Cl (pH 8.0), 20% Glycerol, 100 mM KCl, 1 mM DTT and 0.2 mM EDTA. The extract is stored at -80oC. Avoid repeated freezing and thawing cycles. | | Applications | The HeLa cell nuclear extract of this catalog number is specifically recommended for: 1) in vitro transcription; 2) protein-DNA/RNA and protein-protein interactions; and 3) as a source of individual transcription factors. Although this extract also contains most pre-mRNA processing factors, it is, however, not recommended for in vitro pre-mRNA processing. For Research Use Only. | | References | 1. Dignam, J.D., et al., (1983) Nucleic Acids Res. 11, 1475-1489
2. Manley, J.L., et al., (1980) Proc. Natl. Acad. Sci. USA 77, 5706-5710
3. Sawadogo, M., et al., (1985) Proc. Natl. Acad. Sci. USA 82, 4394-4398
4. Kadonaga, J.T., et al., (1986) Proc. Natl. Acad. Sci. USA 83, 5889-5893
5. Reinberg, D., et al., (1987) J. Biol. Chem. 262, 3322-3330
6. Horikoshi, M., et al., (1988) Cell 54, 1033-1042
7. Yang, X.J., et al., (1996) Nature 382, 19-24
8. Hoeffler, W.K., et al., (1988) Cell 53, 907-920
9. Ge, H., et al., (1996) Methods Enzymol. 274, 57-71
10. Maldonado, E., et al., (1996) Methods Enzymol. 274, 72-100 |
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