Preparation And Storage
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- Triton is a trademark of the Dow Chemical Company.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
ATM is the gene that is mutated in ataxia telangiectasia, an autosomal recessive hereditary disease that is characterized by delayed motor and sexual development, weakened immunity, multiple skin changes, increased sensitivity to ionizing radiation, and increased risk of developing certain cancers. The protein encoded by ATM is a phophoinositide-3-kinase-related protein kinase that is found in the nucleus and is responsible for early responses to the changes in chromatin structure caused by DNA double-strand breaks. Inactive wild type ATM protein forms homodimers or higher order multimers in which the C-terminal kinase domains are inhibited. It has been proposed that multimeric ATM is recruited to DNA double-strand breaks through interactions between its N-terminal HEAT domains and specific chromatin protein complexes. The inhibition of the kinase domains is lifted, resulting in auto-phosphorylation of the ATM at a single serine site and disruption of the multimers. The phosphorylated state of ATM is a rapid and highly sensitive indicator that cells have been exposed to agents that cause DNA double-strand breaks. The kinase domains of the resulting ATM monomers are accessible to a wide variety of substrates that are involved in DNA repair, cell-cycle regulation, and apoptosis. Thus ATM is a central regulator of cellular responses to ionizing radiation, and cells that lack ATM undergo radioresistant DNA synthesis and are resistant to γ radiation-induced apoptosis.
The K88-534 monoclonal antibody recognizes human ATM phosphorylated at serine 1981 (S1981) and mouse ATM phosphorylated at its orthologous site, S1987.
Development References (3)
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Bakkenis CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature. 2003; 421:499-506. (Biology).
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Pellegrini M, Celeste A, Difilippantonio S, et al. Autophosphorylation at serine 1987 is dipensable for murine Atm activation in vivo. Nature. 2006; 443:222-225. (Clone-specific: Western blot).
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You Z, Chahwan C, Bailis J, Hunter T, Russell P. ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Mol Cell Biol. 2005; 25(13):5363-5379. (Biology).
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.