The S100 cytoplasmic extract (S100) is specifically recommended for: 1) in vitro splicing; 2) protein-DNA/RNA and protein-protein interactions; and 3) source of individual splicing factors and other regulatory proteins. Extract activity validated using an in vitro pre-mRNA splicing assay in the presence of SF2/ASF.

The development of cell-free systems that accurately process pre-mRNA by splicing, polyadenylation and editing has led to insights into the mechanisms and the biochemical characteristics of these reactions (1-3). Splicing of pre-mRNA requires the presence of specific sequence elements (cis factors) as well as many protein factors. Although the splicing reaction proceeds in the nucleus, many splicing factors have been found existing in cell cytoplasm. Most essential splicing factors including snRNPs, hnRNPs and debranching enzymes are indeed present in HeLa cell cytoplasmic extract (4-6) and sufficient to support pre-mRNA splicing in vitro when complemented with either naturally purified or recombinant essential splicing factor SF2/ASF (7-9). In addition, some transcription factors for RNA polymerase II, such as TFIIH, are also present in HeLa cytosol extract.

Source
The S100 cytoplasmic extract is specifically recommended for 1) in vitro splicing; 2) protein-DNA/RNA and protein-protein interactions; and 3) source of individual splicing factors and other regulatory proteins.

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 in the presence of SF2/ASF (cat.#P3001).

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
Extract activity validated using an in vitro pre-mRNA splicing assay in the presence of SF2/ASF (cat.#P3001). 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.

References
1. Padgett, R.A. et al., (1983) Proc. Natl. Acad. Sci. USA 80, 5230-5234
2. Krainer, A.R. et al., (1984) Cell 36, 993-1005
3. Manely, J.L. (1984) Cell 33, 595-605
4. Ruskin, B. et al., (1995) Science 229, 135-140
5. Kramer, A. et al., (1985) EMBO J. 4, 3571-3581
6. Krainer and Maniatis (1985) Cell 42, 725-736
7. Krainer, A.R. et al., (1990) Genes Dev. 4, 1158-1171
8. Ge, H. et al., (1991) Cell 66, 373-382
9. Mayeda, A.K. et al., (1999) Methods Mol. Biol. 118, 309-314

DISCLAIMER

This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.