一般描述
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.
免疫原
Human
a synthetic peptide (CKSLASFIKKPFDRGEVESMEDDTNG) corresponding to amino acids 819-844 of human ATM
应用
Immunoblotting (2 µg/ml, see comments)
Immunoprecipitation (2 µg)
Immunoprecipitation (2 µg)
包装
Please refer to vial label for lot-specific concentration.
外形
In 50 mM sodium phosphate buffer, 0.2% gelatin.
分析说明
Negative Control
GM02052A cells
GM02052A cells
Positive Control
Daudi or HeLa cells
Daudi or HeLa cells
其他说明
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).
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.
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.
法律信息
CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
免责声明
Toxicity: Standard Handling (A)
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储存分类代码
10 - Combustible liquids
WGK
nwg
闪点(°F)
Not applicable
闪点(°C)
Not applicable
S Faderl et al.
Leukemia, 16(6), 1045-1052 (2002-06-01)
It has been suggested that the expansion of the leukemic cells in chronic lymphocytic leukemia (CLL) is due to dysregulation of pathways of programmed cell death (apoptosis) rather than cell proliferation, although differences may exist in early vs late and
Fengxia Du et al.
Biochemical and biophysical research communications, 452(4), 1034-1039 (2014-09-23)
The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer
Michael G Kemp et al.
The Journal of biological chemistry, 286(22), 19237-19246 (2011-04-14)
A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro
Xiaofeng Jiang et al.
The Journal of biological chemistry, 281(23), 15741-15746 (2006-04-11)
Members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including the ATM, DNA-PKcs, Atr, and Trrap proteins, function in signal transduction pathways that activate the DNA damage response. PIKK proteins contain a conserved C-terminal FAT/kinase domain/FATC domain structure. The FATC domain
Ye Xu et al.
The Journal of cell biology, 191(1), 31-43 (2010-09-30)
The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair.
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