Specification Please note DAKO Sheet Some information s Lot dependent Monoclonal mouse uman p Clone SX118 Code No M 7202 Lot 088 Edition 09.11.00 Introduction p21 is a protein encoded by the WAF1/Cip1 gene on chromosome 6p. p21 Ar p has been shown to inhibit the activity of several cyclin/cyclin-dependent kinase complexes and to block cell cycle progression (1). It was identified as a gene whose product was transcriptionally activated by wild-type but not mutant p53 protein, serving as a mediator of the cell cycle arrest function of p53( 2 ). In tumour cells that have lost the p53 protein or contain an altered form of p53, p21 levels are dramatically reduced or totally absent(2) This could lead to the passing of the G1 checkpoint, permitting the progression of the cell and oncogenesis. Because p21 appears to mediate several of the growth-reguatapl cycle in the presence of dNa alterations and in this way may result in genomic instability functions of p53, its expression would be predicted to reflect the functional status of p53 more precisely than p53 accumulation. The p21 AF l/cip protein can also be induced in a p53-independent manner and has been shown to play a role in differentiation (3) Furthermore, p21 r p can inhibit DNA replication by blocking the action of proliferating ell nuclear antigen(PCNA)(4). The expression of p21 has been studied immunohistochemistry in a wide range of human tumours including melanomas(5) pancreatic carcinomas(6), cervical carcinomas(7), thymomas(8), thyroid carcinomas( 9) breast carcinomas(10), head-and-neck carcinomas(11), colon carcinomas(12) and Hodgkins disease(13) Presentation Monoclonal mouse antibody supplied in liquid form as tissue culture supernatant(RPMI 1640 medium containing fetal calf serum) dialysed against 0.05 mol/L Tris/HCL, pH 7.2 containing 15 mmol/L NaN3 Total protein concentration: 11.1 g/L Storage 2-8°. Clone sx118.(14) Immunogen Purified glutathione S-transferase GST)-P27 WAF 1/Clp1 fusion protein Specificitylreactivity The antibody reacts with a 21 kDa protein in immunoblotting of MCF-7 cells and immunoprecipitates a 21 kDa band from lysates from SAOS-2 cells transfected with the full length p21 Ar cp protein(14). The antibody also reacts with p21 from mouse Application The antibody can be used in immunohistochemistry. Furthermore, the antibody produced by the clone SX118 has been shown to be applicable for immunoblotting an immunoprecipitation(14) Staining procedures Formalin-fixed and paraffin-embedded sections Can be used on formalin-fixed, paraffin-embedded tissue sections. Antigen retrieval, such as by heating in 10 mmol/L citrate buffer or in DAKO Target Retrieval Solution, code No. S1700, is mandatory. The slides should not be allowed to dry out during this treatment or during the following immunohistochemical staining procedure
Specification Sheet Please note Some information is Lot dependent. Monoclonal Mouse Anti-Human p21WAF1/Cip1 Clone SX118 Code No. M 7202 Lot 088. Edition 09.11.00 Introduction p21 is a protein encoded by the WAF1/Cip1 gene on chromosome 6p. p21WAF1/Cip1 has been shown to inhibit the activity of several cyclin/cyclin-dependent kinase complexes and to block cell cycle progression (1). It was identified as a gene whose product was transcriptionally activated by wild-type but not mutant p53 protein, serving as a mediator of the cell cycle arrest function of p53 (2). In tumour cells that have lost the p53 protein or contain an altered form of p53, p21 levels are dramatically reduced or totally absent (2). This could lead to the passing of the G1 checkpoint, permitting the progression of the cell cycle in the presence of DNA alterations and in this way may result in genomic instability and oncogenesis. Because p21 appears to mediate several of the growth-regulatory functions of p53, its expression would be predicted to reflect the functional status of p53 more precisely than p53 accumulation. The p21WAF1/Cip1 protein can also be induced in a p53-independent manner and has been shown to play a role in differentiation (3). Furthermore, p21WAF1/Cip1 can inhibit DNA replication by blocking the action of proliferating cell nuclear antigen (PCNA) (4). The expression of p21WAF1/Cip1 has been studied by immunohistochemistry in a wide range of human tumours including melanomas (5), pancreatic carcinomas (6), cervical carcinomas (7), thymomas (8), thyroid carcinomas (9), breast carcinomas (10), head-and-neck carcinomas (11), colon carcinomas (12) and Hodgkin’s disease (13). Presentation Monoclonal mouse antibody supplied in liquid form as tissue culture supernatant (RPMI 1640 medium containing fetal calf serum) dialysed against 0.05 mol/L Tris/HCl, pH 7.2 containing 15 mmol/L NaN3. Mouse Ig concentration: 390 mg/L. Isotype: IgG1, kappa. Total protein concentration: 11.1 g/L. Storage 2 - 8 °C. Clone SX118. (14). Immunogen Purified glutathione S-transferase (GST)-p21WAF1/Cip1 fusion protein. Specificity/reactivity The antibody reacts with a 21 kDa protein in immunoblotting of MCF-7 cells and immunoprecipitates a 21 kDa band from lysates from SAOS-2 cells transfected with the full length p21WAF1/Cip1 protein (14). The antibody also reacts with p21 from mouse. Application The antibody can be used in immunohistochemistry. Furthermore, the antibody produced by the clone SX118 has been shown to be applicable for immunoblotting and immunoprecipitation (14). Staining procedures Formalin-fixed and paraffin-embedded sections Can be used on formalin-fixed, paraffin-embedded tissue sections. Antigen retrieval, such as by heating in 10 mmol/L citrate buffer, pH 6.0, or in DAKO Target Retrieval Solution, code No. S 1700, is mandatory. The slides should not be allowed to dry out during this treatment or during the following immunohistochemical staining procedure. (2)
For tissue sections sensitive staining techniques are recommended, such as the LSABO+or the EnVision M+ systems The antibody may be used at a dilution of 1: 25-1: 50 with the LSAB@+ system when tested on formalin-fixed, paraffin-embedded sections of human tonsil Frozen sections and cell smears Can be used for labelling acetone-fixed cryostat sections or for fixed cell smears. For staining cell smears, the APAAP method is recommended The antibody may be used at a dilution at 1: 25-1: 50 in the APAAP technique, when tested on acetone-fixed cryostat sections of human tonsil These are guidelines only, optimal dilutions should be determined by the individual laborator Automation: The antibody can be used on automated immunostaining systems References (1) Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D p21 is a universal inhibitor of cyclin kinases. Nature 1993: 366: 701-4 (2) El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression Cell 1993: 75: 817-25 (3) Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, El-Houseini ME. Induction of p21 (WAF. 1/CIP1)during differentiation. Oncogene 1994: 9: 3389-96 (4) Li R, Waga S, Hannon GJ, Beach D, Stillman B Differential effects by the p21 CDK inhibitor on PCNA- dependent DNA replication and repair. Nature 1994: 371: 534-7 (5)Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi MC, Holbrook NJ. p21 afl/cipl protects against p53- mediated apoptosis of human melanoma cells. Oncogene 1997: 14: 929-3 ham sT, Dugan MC, Joshi US, Chen YC, mith DW, et al. The clinical significance of nd p53 expression in pancreatic adenocarcinoma Cancer 1997: 80: 372-81 (7) Palazzo JP, Mercer WE, Kovatich AJ, McHugh M. Immunohistochemical localization of p21 AF1CP1 in normal, hyperplastic, and neoplastic uterine tissues. Hum Pathol 1997: 27: 60-6 (8) Stefanaki K, Rontogianni D, Kouvidou CH, Bolioti S, Delides G, Pantelidaki A, et al. Expression of p53 mdm2, p21/waf1 and bcl-2 proteins in thymomas. Histopathol 1997: 30: 549-55 (9) Ito Y, Kobayashi T, Takeda T, Komoike Y, Wakasugi E, Tamaki Y, et al. Expression of p21 (WAF 1/CIP1) rotein in clinical thyroid tissues. Br J Cancer 1996: 74: 1269-74 genotoxic agents in wild-type TP53 expressing breast primary tumours. Oncogene 1997; 14. 45-Donse to (10) Guillot C, Falette N, Paperin M-P, Courtois S, Gentil-Perret A, Treilleux l et al. p21 WAFlcP'resp (11)Erber R, Klein W, AndI T, Enders C, Born Al, Conradt C, et al. Aberrant p21 P1/AF1 protein accumulation head-and-neck cancer. Int J Cancer 1997: 74: 383-9 (12)Valassiadou KE, Stefanaki K, Tzar Datseris G, Georgoulias V, Melissas J,et al Immunohistochemical expression of p53, bcl-2, mdm2 adenocarcinomas Anticancer Res 1997: 17: 2571-6 (13)Naresh KN, O'Conor GT, Soman CS, Johnson J, Advani SH, Magrath IT, et al. a study of p53 protein odgkin's disease at presentation and relapse 1997;28:54955 (14)Fredersdorf S, Milne AW, Hall PA, Lu X Characterization of a panel of novel anti-p21 wati/ monoclonal antibodies and immunochemical analysis of p21 expression in nomal human tissues. Am J Pathol1996;148:825-35 M7202JA09.1100
- 2 - For tissue sections sensitive staining techniques are recommended, such as the LSAB+ or the EnVision™+ systems. The antibody may be used at a dilution of 1:25 - 1:50 with the LSAB+ system when tested on formalin-fixed, paraffin-embedded sections of human tonsil. Frozen sections and cell smears Can be used for labelling acetone-fixed cryostat sections or for fixed cell smears. For staining cell smears, the APAAP method is recommended. The antibody may be used at a dilution at 1:25 - 1:50 in the APAAP technique, when tested on acetone-fixed cryostat sections of human tonsil. These are guidelines only; optimal dilutions should be determined by the individual laboratory. Automation: The antibody can be used on automated immunostaining systems. References (1) Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993;366:701-4. (2) El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817-25. (3) Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, El-Houseini ME. Induction of p21 (WAF- 1/CIP1) during differentiation. Oncogene 1994;9:3389-96. (4) Li R, Waga S, Hannon GJ, Beach D, Stillman B. Differential effects by the p21 CDK inhibitor on PCNAdependent DNA replication and repair. Nature 1994;371:534-7. (5) Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi MC, Holbrook NJ. p21Waf1/Cip1 protects against p53- mediated apoptosis of human melanoma cells. Oncogene 1997;14:929-35. (6) Dergham ST, Dugan MC, Joshi US, Chen YC, Du W, Smith DW, et al. The clinical significance of p21WAF1/CIP1 and p53 expression in pancreatic adenocarcinoma. Cancer 1997;80:372-81. (7) Palazzo JP, Mercer WE, Kovatich AJ, McHugh M. Immunohistochemical localization of p21WAF1/CIP1 in normal, hyperplastic, and neoplastic uterine tissues. Hum Pathol 1997;27:60-6. (8) Stefanaki K, Rontogianni D, Kouvidou CH, Bolioti S, Delides G, Pantelidaki A, et al. Expression of p53, mdm2, p21/waf1 and bcl-2 proteins in thymomas. Histopathol 1997;30:549-55. (9) Ito Y, Kobayashi T, Takeda T, Komoike Y, Wakasugi E, Tamaki Y, et al. Expression of p21 (WAF1/CIP1) protein in clinical thyroid tissues. Br J Cancer 1996;74:1269-74. (10) Guillot C, Falette N, Paperin M-P, Courtois S, Gentil-Perret A, Treilleux I, et al. p21 WAF1/CIP1 response to genotoxic agents in wild-type TP53 expressing breast primary tumours. Oncogene 1997;14:45-52. (11) Erber R, Klein W, Andl T, Enders C, Born AI, Conradt C, et al. Aberrant p21CIP1/WAF1 protein accumulation in head-and-neck cancer. Int J Cancer 1997;74:383-9. (12) Valassiadou KE, Stefanaki K, Tzardi M, Datseris G, Georgoulias V, Melissas J, et al. Immunohistochemical expression of p53, bcl-2, mdm2 and waf1/p21 proteins in colorectal adenocarcinomas. Anticancer Res 1997;17:2571-6. (13) Naresh KN, O’Conor GT, Soman CS, Johnson J, Advani SH, Magrath IT, et al. A study of p53 protein, proliferating cell nuclear antigen, and p21 in Hodgkin’s disease at presentation and relapse. Hum Pathol 1997;28:549-55. (14) Fredersdorf S, Milne AW, Hall PA, Lu X. Characterization of a panel of novel anti-p21Waf1/Cip1 monoclonal antibodies and immunochemical analysis of p21Waf1/Cip1 expression in normal human tissues. Am J Pathol 1996;148:825-35. M 7202/JA/09.11.00
