usage
vial sufficient for 50 reactions
shipped in
dry ice
storage temp.
−70°C
Application
A cell-free transcription system that produces accurate transcripts with RNA polymeraseII. Reaction will incorporate 50-150fmol nucleotides into a 400-nucleotide transcript from Ad2ML (adenovirus major late promoter) in 60min at 30°C.
Biochem/physiol Actions
The system carries out basal transcription, dependent on recognition of the TATA element, as well as transcription which is regulated by interaction of transcription factors with upstream promoter elements, e.g., USF, Oct-1, Sp-1, progesterone receptor.
Depleting the extract of individual transcription factors produces a system for assaying purified factors (TATA binding protein, Oct-1, etc.) by complementation. The extract is also a source of transcriptional regulators and RNA processing proteins.
Depleting the extract of individual transcription factors produces a system for assaying purified factors (TATA binding protein, Oct-1, etc.) by complementation. The extract is also a source of transcriptional regulators and RNA processing proteins.
Packaging
Supplied in 20 mM HEPES, pH 9.0, containing 100 mM KCl, 0.2 mM EDTA, 1 mM DTT, and 20% glycerol.
Analysis Note
The extract has been treated with PMSF to inhibit proteases
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Sawadogo, M., et al.
The Journal of Biological Chemistry, 263, 11985-11985 (1985)
Briggs, M.R., et al.
Science, 243, 47-47 (1986)
R W Carthew et al.
Cell, 43(2 Pt 1), 439-448 (1985-12-01)
A gel electrophoresis DNA binding assay has been used to identify proteins in HeLa cell extracts that specifically bind to the major late promoter of adenovirus. A major late promoter transcription factor MLTF has been detected as a discrete protein-DNA
A R Krainer et al.
Cell, 36(4), 993-1005 (1984-04-01)
Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by
T M Kristie et al.
The EMBO journal, 8(13), 4229-4238 (1989-12-20)
The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation
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