ATM Antibody

What are the primary applications of ATM antibodies in experimental research?

ATM antibodies are widely used to investigate the protein’s role in DNA repair, cell cycle regulation, and cancer pathogenesis. Key applications include:

• Immunohistochemistry (IHC): Detecting ATM expression patterns in formalin-fixed, paraffin-embedded (FFPE) tissues, such as gastric cancer (negative staining) and breast/lung cancers (low expression linked to poor prognosis) .

• Western Blot (WB): Identifying full-length ATM (~350 kDa) and its splice variants in cell lysates (e.g., HEK-293, HeLa, MCF-7 cells) .

• Immunofluorescence (IF): Localizing ATM in nuclear compartments during DNA damage responses, as demonstrated in HepG2 and HeLa cells .

For IHC, antigen retrieval methods vary by tissue type. For example, mouse brain tissues require TE buffer (pH 9.0), while human breast cancer tissues may perform better with citrate buffer (pH 6.0) .

How should researchers validate ATM antibody specificity in immunohistochemistry?

Antibody validation requires a multi-step approach:

• Positive/Negative Controls: Include tissues with known ATM expression (e.g., fallopian tube, brain) and ATM-deficient samples (e.g., Bal/Bal mouse brain) .

• Preabsorption Tests: Preincubate antibodies with immunizing peptides to confirm signal loss, as shown in human cerebellar neurons .

• Cross-Verification: Compare results across clones (e.g., EP327 , 83608-5-RR ) and methodologies (IHC vs. WB).

A study using the 2C1A1 antibody in Awb/Awb mice revealed residual ATM peptides via mass spectrometry, underscoring the importance of orthogonal validation .

How can conflicting results in ATM protein detection across studies be resolved?

Discrepancies often arise from tissue-specific splicing variants or epitope accessibility. For example:

• Mouse Models: Bal/Bal mice show no ATM protein despite normal mRNA levels, whereas Awb/Awb mice produce truncated but detectable ATM .

• Antibody Epitopes: The 2C1A1 antibody detects ATM in human A-T Purkinje cells, while Y-170 (targeting phosphorylated serine 1981) does not, suggesting epitope masking in mutants .

To resolve conflicts, researchers should:

• Use multiple antibodies targeting distinct epitopes.

• Validate findings with functional assays (e.g., kinase activity tests) .

What methodological considerations are critical when analyzing ATM phosphorylation status in DNA damage response studies?

ATM activation involves autophosphorylation at serine 1981 (S1981), but technical challenges include:

• Antibody Specificity: Phospho-S1981 antibodies may fail to recognize ATM in certain mutants (e.g., Awb/Awb mice) . Alternative epitopes like S1987 are more reliable in some contexts .

• Co-Immunoprecipitation (Co-IP): Confirm ATM’s interaction with partners like MDC1 post-irradiation. A 10 Gy γ-ray dose induces ATM-MDC1 binding, which is abolished in S1981A mutants .

How do tissue-specific factors influence ATM antibody performance in preclinical models?

Brain tissues pose unique challenges due to alternative splicing and truncated ATM isoforms:

• Mouse Cerebellum: Bal/Bal Purkinje cells exhibit cell cycle reentry despite ATM mRNA presence, necessitating functional assays (e.g., BrdU incorporation) .

• Human A-T Brains: Retained 2C1A1 immunoreactivity in Purkinje cells suggests stable mutant ATM production, complicating phenotype-genotype correlations .

For neuronal studies, combine IHC with RT-PCR to assess mRNA-protein discordance .