| Purification and Quality Control | The Flag-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified WT-1 is greater than 90% homogeneous based on SDS-PAGE analysis. | | Unit Definition (Activity) | 1 unit equals 1 nanogram of purified protein. 1 unit is the amount sufficient for a gel mobility shift assay in a 20 µl reaction. 50 units are sufficient for reconstituted transcription assay and 100 units are sufficient for a protein-protein interaction assay. | | Applications | Recombinant WT-1 protein can be used for: 1) in vitro function studies including transcription, DNA or RNA binding assays; 2) protein-protein interaction assay; and 3) cell growth and proliferation assays. | | 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. | | Background | WT-1, the product of Wilms’ tumor suppressor gene Wt1, is a nuclear protein with structural motifs characteristic of transcription factors, including four C-terminal zinc fingers. While different pre-mRNA processing could result in 16 isoforms of the protein, the inclusion or exclusion of exon 5 and three amino acids (KTS) between zinc fingers 3 and 4 largely affects the activity of the WT1 protein (1-3). Such a complex post-transcriptional regulation, particularly in splicing, may represent a major regulatory mechanism for tumorigenesis of the Wilms’ tumor. With the inclusion of exon 5, WT1 (KTS+) binds to both DNA and RNA and is RNase but not DNase sensitive (4). This form also co-localizes with splicing factors in a speckled nuclear particle, suggesting that the WT1 protein may function as both a transcription factor and a splicing regulator (5, 6). | | References | 1. Haber, DA. et al., (1991) Proc. Natl. Acad. Sci. USA 88, 9618-9622; (1993) Science 262, 2057-2059
2. Kreidberg, JA. et al., (1993) Cell 74, 679-691
3. Larsson, SH. et al., (1995) Cell 81, 391-401
4. Caricasole, A., et al., (1996) Proc. Natl. Acad. Sci. USA, 93, 7562-7566
5. Little, M. et al., (1997) Hum. Mutat. 9, 209-225
6. Englert, C. et al., (1995) EMBO J. 14, 4662-4675 |
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