Description General description Anti-ATM (Ab-3) (819-844), rabbit polyclonal, recognizes the ~350 kDa ATM protein in Daudi and HeLa cells. It is validated for Western blotting and immunoprecipitation. Purified rabbit polyclonal antibody. Recognizes the ~350 kDa ATM protein. Recognizes the ~350 kDa ATM protein in Daudi and HeLa cells. Immunogen Human a synthetic peptide (CKSLASFIKKPFDRGEVESMEDDTNG) corresponding to amino acids 819-844 of human ATM Application Immunoblotting (2 µg/ml, see comments) Immunoprecipitation (2 µg) Physical form In 50 mM sodium phosphate buffer, 0.2% gelatin. Packaging 100 μg in Plastic ampoule Please refer to vial label for lot-specific concentration. Warning Toxicity: Standard Handling (A) Analysis Note Negative Control GM02052A cells Positive Control Daudi or HeLa cells Other Notes For immunoblotting use 100-150 µg cell lysate on a 5% acrylamide gel (SDS/PAGE) and transferred to nitrocellulose using semi-dry transfer at 9V constant voltage for 2 h. Detection of antibody/antigen complexes is done using HRP conjugated goat anti-rabbit IgG at 25 ng/ml (Cat. No. DC03L). 1. Anti-ATM (Ab-3) Rabbit pAb immunoblots and immunoprecipitates a 350 kDa protein in lysates of normal and transformed cells or tumor lines derived from individuals homozygous wild type for the ATM gene; whereas in cells derived from patients with Ataxia-Telangiectasia and carrying homozygous inactivating mutations in ATM, no 350 kDa protein is detected. 2. Anti-ATM (Ab-3) Rabbit pAb may non-specifically detect smaller molecular weight proteins present in both ATM mutant and wild type cells. Careful titering of primary and secondary antibodies is recommended. 3. Immunoblotting of p350ATM requires loading 100-150 µg cell lysate on low percentage acrylamide gels (5%) (SDS/PAGE) with electrophoresis performed until the 200 kDa molecular weight marker has migrated halfway through the gel. Semi-dry electrophoretic transfer is for 2 h at 9V constant voltage. Tank transfer is overnight at 40 V constant voltage. 4. To transfer the gel for blotting, lay a dry piece of Whatman 3MM Chromatography paper over the wet gel. Carefully peel the 3MM paper and gel off the glass plate and immerse, gel side up, in transfer buffer until 3MM paper is thoroughly wet. Remove bubbles by rolling a pipette across the surface of the gel. 5. To confirm detection of p350ATM, a cell line carrying a truncating mutation in ATM should be used as a negative control. Cell lines from A-T patients which show no detectable band at 350 kDa, can be obtained from the National Institute of General Medical Sciences Human Genetic Mutant Cell Repository at the Coriell Institute for Medical Research, 401 Haddon Avenue, Camden, NJ 08103. 6. For immunoprecipitations, prepare nuclear lysates as described. Immunoprecipitate p350ATM using 2 µg of Anti-ATM (Ab-3) Rabbit pAb and Protein A-Agarose (Cat. No. IP06). Detection can be after metabolic labeling with 35S methionine followed by autoradiography, or alternatively, immunoprecipitated proteins can be displayed on 5% SDS/PAGE, transferred to nitrocellulose and then blotted as above using Anti-ATM (Ab-3). Friedberg, E.C., et al. 1995. Amer. Soc. of Microbiolgy (meeting report), Wash. D.C. Meyn, S.M. 1995. Cancer Res.55, 5991. Paules, R.S., et al. 1995. Cancer Res.55, 1763. Savitsky, K., et al. 1995. Science268, 1749. Savitsky, K., et al. 1995. Hum. Molec. Genet.4, 2025. Zakian, V., 1995. Cell82, 685. Beamish, H., et al. 1993. Rad. Res.138, 130. Kastan, M.B., et al. 1992. Cell71, 587. Painter, R.B. and Young, B.R. 1980. Proc. Natl. Acad. Sci. USA77, 7315.