Mammalian Cell Extract Technology
After years of research and development, AscentGene is proud to offer high-quality active cell extracts, isolated from both the cytosol and nucleus. These unique extracts are applicable in a wide variety of functional assays in vitro, such as:
- Pre-mRNA splicing assays
- Polyadenylation assays
- Transcription assays
- Translation assays
- Kinase assays
- Gel shift assays (protein-DNA/RNA interactions)
- Protein-protein interaction assays
- Western blot assays
Additionally, these active nuclear and cytoplasm extracts can be used for native protein isolation and identification, specifically as sources of individual cancer biomarkers, tumor suppressors, kinases, cytokines, splicing factors, polyadenylation factors, transcription factors, ubiquitylation/SUMOylation factors, discovery and characterization of disease-related new proteins, protein expression profiles, and protein location studies.
In vitro transcription of HIV promoter-containing DNA template with 293 (lanes 1 and 2) or HeLa (lanes 3 and 4) nuclear extract (Catalog # AG1011 and AG1012) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of activator Gal4-AH and the coactivator PC4.
In vitro splicing of SV40 early pre-mRNA using HeLa (lanes 1-5) and 293 (lanes 6-10) nuclear extracts (Catalog # AG1012 and AG1011) does not require the presence of additional KCl.
The native Sp1 protein purified from HeLa cell nuclear extract (Catalog # AG1012) is functional and shows the expected specific DNA binding activity in Gel Shift Assay. The protein is active in in vitro transcription assays.
The native casein kinase II (nCKII) purified from HeLa cell nuclear extract (Catalog # AG1012) reveals similar activity in Kinase Assay, as compared to recombinant CKII (rCKII).
References
Manley, J.L., Sharp, P.A. and Gefter, M.L. (1982) RNA synthesis in isolated nuclei processing of adenovirus serotype 2 late messenger RNA precursors. J. Mol. Biol. 159, 581-99.
Ge, H. and Manley, J.L. (1990) A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell 62, 25-34.
Ge, H., Zuo, P. and Manley, J.L. (1991) Primary structure of the human splicing factor ASF reveals similarities with Drosophilia regulators. Cell 66, 373-82.
Dignam J.D., Lebovitz R.M., Roeder R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475-89.
Ge, H. and Roeder, R.G. (1994) Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes. Cell 78, 513-23.
Ge, H., Martinez, E., Chiang, C.M. and Roeder, R.G. (1996) Activator-dependent transcription factors and cofactors. Methods in Enzymology 274, 570-71.