Description General description Recognizes the ~21 kDa p21WAF1 protein in skin and colon tissue and in cells expressing wild-type p53 (e.g. Hs27 or U205 cells treated with DNA damaging agents). This Anti-p21WAF1 (Ab-1) Mouse mAb (EA10) is validated for use in FC, Immunoblotting, IF, IP, Paraffin Sections for the detection of p21WAF1 (Ab-1). Application Flow Cytometry (2 µg/ml or use Cat. No. OP64F; see application references) Frozen Sections (5 µg/ml or use Cat. No. OP64F) Immunoblotting (1-3 µg/ml) Immunofluorescence (1-5 µg/ml or use Cat. No. OP64F) Immunoprecipitation (2 µg/sample) Paraffin Sections (5 µg/ml or use OP64F; heat pre-treatment required; see comments and application references) Packaging 20, 100 μg in Plastic ampoule Please refer to vial label for lot-specific concentration. Warning Toxicity: Standard Handling (A) Analysis Note Positive Control Any cell line expressing wild-type p53 (e.g. Hs27 or U2OS treated with DNA-damaging agents) or skin or colon tissue Other Notes Agarwal, M.L., et al. 1995. Proc. Natl. Acad. Sci. USA92, 8493. Chen, Y.Q., et al. 1995. Int. J. Oncology7, 889. Deng, C., et al. 1995. Cell82, 675. El-Deiry, W.S., et al. 1995. Cancer Res.55, 2910. Waldman, T., et al. 1995. Cancer Res.55, 5187. Elbendary, A., et al.1994. Cell Growth Diff.5, 1301. El-Deiry, W.S., et al. 1994 Cancer Res.54, 1169. Li, R., et al. 1994. Nature371, 534. Michieli, P., et al. 1994. Cancer Res.54, 3391. Noda, A., et al.1994. Exp. Cell Res.211, 90. El-Deiry, W.S., et al.1993. Cell75, 817. Gu, Y., et al. 1993. Nature366, 707. Harper, J.W., et al.1993. Cell75, 805. Xiong, Y., et al.1993. Genes Devel.7, 1572. Xiong, Y., et al.1993. Nature366, 701. Xiong, Y., et al.1992. Cell71, 505. Maximal p21WAF1 expression requires wild type p53 activity. Treatment of U2OS or MCF7 cells with DNA damaging agents (such as doxorubicin at 0.2 µg/ml) induces wild type p53 expression which in turn activates WAF1 expression. Serum stimulation of quiescent cells will give low level WAF1 expression independent of p53 expression. Untreated cells will express little p21WAF1 and can be used as a negative control. Cat. No. OP64F was tested in HALT cells induced by incubation at 31°C; FITC-goat anti-mouse IgG (Cat. No. DC13L) was used as a negative control. This antibody will immunoprecipitate p21WAF1 but not associated proteins. For immunoblotting applications, use a 0.22 µm filter and visualize by chemiluminescence. For staining paraffin sections, heating the tissue in 10 mM citrate buffer is required (see application references). In either paraffin or frozen sections of normal human colon, the non-dividing cells of colonic epithelium will stain positive for p21WAF1 while the proliferating compartment of crypts will not stain. Antibody should be titrated for optimal results in individual systems.