Native TFIIH is multicomponent basal transcription factor complex and purified from hela nuclear extract.
Purification and Quality Control
Native TFIIH complex is isolated from HeLa nuclear extract after several chromatographic purification steps.
Unit Definition (Activity)
1 unit equals 1 nanogram of purified protein. 100 units are sufficient for a reconstituted in vitro transcription assay and 500 units are sufficient for a protein-protein interaction assay detected by immunoblot.
Applications
Purified TFIIH has been applied for in vitro transcription assays, DNA repair, DNA-protein, RNA-protein, protein-protein interaction assays as well as for in vitro correction of the nuclear excision repair defect of XPD, XPB and TTD-A fibroblasts (12). The TFIIH protein complex is 60%-70% pure and is devoid of other general transcription factors. For Research use only.
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.
TFIIH is a multicomponent basal transcription factor complex. Nine subunits have been identified within the TFIIH holoenzyme complex (1, 2). Various enzymatic activities, including DNA repair (3), helicase (4), and cyclin-dependent kinase (5) activities, have been reported. The XPB, p62, p52, p44, and p34 subunits are thought to constitute the "core" of the TFIIH transcription machinery (6). Although the p44 and p34 subunits have no defined enzymatic activity, their zinc finger structures suggest that they may be DNA-binding proteins (7) that might mediate interactions with soluble transcription factors. The cdk-activating kinase (CAK) subcomplex, comprising subunits Cdk7, cyclin H, amd MAT1, phosphorylate several cyclin-dependent kinases (cdks), as well as the carboxy-terminal domain of pol II (1, 8). Several inherited human disorders such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) are associated with mutations in TFIIH subunits (9-11).
References
1. Feaver, W.J., et al., (1991) J. Biol. Chem. 266, 19000-19005 2. Flores, O., et al., (1992) J. Biol. Chem. 267, 2786-2790 3. Drapkin, R., et al., (1994) Nature 368, 769-772 4. Schaeffer, L., et al., (1993) Science 260, 58-63 5. Adamczewski, J. P., et al.,(1996) EMBO J. 15, 1877-1884 6. Marinoni, J. C., et al., (1997) EMBO J. 16, 1093-1102 7. Humbert, S., et al., (1994) EMBO J. 13, 2393-2398 8. Roy, R., et al., (1994) Cell 79, 1093-1101 9. Taylor, E.M., et al., (1997) Proc. Natl. Acad. Sci. 94, 8658-8663 10. Botta, E., et al., (1998) Am. J. Hum. Genet. 63, 1036-1048 11. de Laat, W.L. et al., (1999) Genes & Dev. 13, 768-785 12. Vermeulen, W. (1994) Cold Spring Harbour Quant. Symp. LIX, 317-329
DISCLAIMER
This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.
