Recombinant Anti-ATM antibody [Y170] (ab32420)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y170] to ATM
- Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-ATM antibody [Y170]
See all ATM primary antibodies -
Description
Rabbit monoclonal [Y170] to ATM -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details
Unsuitable for: IP -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: 293 cell lysate. Flow Cyt (intra): HeLa cells ICC/IF: HeLa and HepG2 cells
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y170 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32420 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) |
1/20.
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WB | (5) |
1/1000 - 1/10000. Predicted molecular weight: 350 kDa.
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IHC-P | (3) |
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
1/250 - 1/500.
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Notes |
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Flow Cyt (Intra)
1/20. |
WB
1/1000 - 1/10000. Predicted molecular weight: 350 kDa. |
IHC-P
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
1/250 - 1/500. |
Target
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Function
Serine/threonine protein kinase which activates checkpoint signaling upon double strand breaks (DSBs), apoptosis and genotoxic stresses such as ionizing ultraviolet A light (UVA), thereby acting as a DNA damage sensor. Recognizes the substrate consensus sequence [ST]-Q. Phosphorylates 'Ser-139' of histone variant H2AX/H2AFX at double strand breaks (DSBs), thereby regulating DNA damage response mechanism. Also plays a role in pre-B cell allelic exclusion, a process leading to expression of a single immunoglobulin heavy chain allele to enforce clonality and monospecific recognition by the B-cell antigen receptor (BCR) expressed on individual B lymphocytes. After the introduction of DNA breaks by the RAG complex on one immunoglobulin allele, acts by mediating a repositioning of the second allele to pericentromeric heterochromatin, preventing accessibility to the RAG complex and recombination of the second allele. Also involved in signal transduction and cell cycle control. May function as a tumor suppressor. Necessary for activation of ABL1 and SAPK. Phosphorylates p53/TP53, FANCD2, NFKBIA, BRCA1, CTIP, nibrin (NBN), TERF1, RAD9 and DCLRE1C. May play a role in vesicle and/or protein transport. Could play a role in T-cell development, gonad and neurological function. Plays a role in replication-dependent histone mRNA degradation. Binds DNA ends. -
Tissue specificity
Found in pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, spleen, thymus, testis, ovary, small intestine, colon and leukocytes. -
Involvement in disease
Defects in ATM are the cause of ataxia telangiectasia (AT) [MIM:208900]; also known as Louis-Bar syndrome, which includes four complementation groups: A, C, D and E. This rare recessive disorder is characterized by progressive cerebellar ataxia, dilation of the blood vessels in the conjunctiva and eyeballs, immunodeficiency, growth retardation and sexual immaturity. AT patients have a strong predisposition to cancer; about 30% of patients develop tumors, particularly lymphomas and leukemias. Cells from affected individuals are highly sensitive to damage by ionizing radiation and resistant to inhibition of DNA synthesis following irradiation.
Note=Defects in ATM contribute to T-cell acute lymphoblastic leukemia (TALL) and T-prolymphocytic leukemia (TPLL). TPLL is characterized by a high white blood cell count, with a predominance of prolymphocytes, marked splenomegaly, lymphadenopathy, skin lesions and serous effusion. The clinical course is highly aggressive, with poor response to chemotherapy and short survival time. TPLL occurs both in adults as a sporadic disease and in younger AT patients.
Note=Defects in ATM contribute to B-cell non-Hodgkin lymphomas (BNHL), including mantle cell lymphoma (MCL).
Note=Defects in ATM contribute to B-cell chronic lymphocytic leukemia (BCLL). BCLL is the commonest form of leukemia in the elderly. It is characterized by the accumulation of mature CD5+ B lymphocytes, lymphadenopathy, immunodeficiency and bone marrow failure. -
Sequence similarities
Belongs to the PI3/PI4-kinase family. ATM subfamily.
Contains 1 FAT domain.
Contains 1 FATC domain.
Contains 1 PI3K/PI4K domain. -
Domain
The FATC domain is required for interaction with KAT5. -
Post-translational
modificationsPhosphorylated by NUAK1/ARK5. Autophosphorylation on Ser-367, Ser-1893, Ser-1981 correlates with DNA damage-mediated activation of the kinase.
Acetylation, on DNA damage, is required for activation of the kinase activity, dimer-monomer transition, and subsequent autophosphorylation on Ser-1981. Acetylated in vitro by KAT5/TIP60. -
Cellular localization
Nucleus. Cytoplasmic vesicle. Primarily nuclear. Found also in endocytic vesicles in association with beta-adaptin. - Information by UniProt
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Database links
- Entrez Gene: 472 Human
- Omim: 607585 Human
- SwissProt: Q13315 Human
- Unigene: 367437 Human
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Alternative names
- A-T mutated antibody
- A-T mutated homolog antibody
- AT mutated antibody
see all
Images
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All lanes : Anti-ATM antibody [Y170] (ab32420) at 1/1000 dilution
Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : ATM knockout MCF7 cell lysate
Lane 3 : Wild-type A549 cell lysate
Lane 4 : ATM knockout A549 ab283811 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 350 kDa
Observed band size: 350 kDaWestern blot: Anti-ATM antibody [Y170] (ab32420) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32420 was shown to bind specifically to ATM. A band was observed at 350 kDa in wild-type MCF7 cell lysates with a reduction in signal observed at this size in ATM heterozygous knockout cell line ab282630. To generate this image, wild-type and ATM heterozygous knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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All lanes : Anti-ATM antibody [Y170] (ab32420) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : ATM knockout A549 cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : U-2 OS cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 350 kDa
Observed band size: 350 kDaFalse colour image of Western blot: Anti-ATM antibody [Y170] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32420 was shown to bind specifically to ATM. A band was observed at 350 kDa in wild-type A549 cell lysates with no signal observed at this size in ATM knockout cell line ab276095 (knockout cell lysate ab283834). To generate this image, wild-type and ATM knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling ATM with ab32420 at 1/500. Goat anti rabbit IgG(Alexa Fluor® 488), ab150077 at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Nulei were counterstained blue with DAPI.
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All lanes : Anti-ATM antibody [Y170] (ab32420) at 1/3000 dilution (Purified)
Lane 1 : HeLa cell lysate
Lane 2 : HEK293 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 350 kDa
Observed band size: 370 kDa why is the actual band size different from the predicted? -
Immunocytochemistry/ Immunofluorescence analysis of HepG2 cells labeling ATM with ab32420 at 1/500. Goat anti rabbit IgG(Alexa Fluor® 488), ab150077 at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Nulei were counterstained blue with DAPI.
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DNA repair proteins accumulate at MVM APAR bodies
Repair proteins accumulate at APAR bodies. NB324K cells were infected with MVMp (MOI of 10) for 16 hr before being fixed and processed for immunofluorescence. Cells were stained with the indicated antibodies to mark DDR repair proteins. APAR bodies were detected with antibodies to NS1. Nuclei were stained with DAPI. All images were captured using an objective of 63×.
Cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 in PBS for 15 minutes.
(Image shows the right-hand panel of Figure 2A)
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Formaldehyde-fixed human colon tissue stained for ATM using ab32420 at 1/100 dilution in immunohistochemical analysis.
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Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling ATM with purified ab32420 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluorr®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
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Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling ATM with ab32420 at 1:100 dilution. Tissue underwent antigen retrieval using Tris/EDTA Buffer (pH9.0). The section was counterstained with haematoxylin.
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Formaldehyde-fixed human serous ovarian tumor tissue stained for ATM using ab32420 at 1/50 dilution in immunohistochemical analysis.
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Immunohistochemical analysis of paraffin-embedded Human normal breast tissue labeling ATM with ab32420 at 1:100 dilution. Tissue underwent antigen retrieval using Tris/EDTA Buffer (pH9.0). The section was counterstained with haematoxylin.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (118)
ab32420 has been referenced in 118 publications.
- El Jabbour T et al. ATM Germline-Mutated Gastroesophageal Junction Adenocarcinomas: Clinical Descriptors, Molecular Characteristics, and Potential Therapeutic Implications. J Natl Cancer Inst 114:761-770 (2022). PubMed: 35078243
- Serafim RB et al. PIMREG expression level predicts glioblastoma patient survival and affects temozolomide resistance and DNA damage response. Biochim Biophys Acta Mol Basis Dis 1868:166382 (2022). PubMed: 35301087
- Zhou ZY et al. Positive regulation of ataxia-telangiectasia-mutated protein (ATM) by E2F transcription Factor 1 (E2F-1) in cisplatin-resistant nasopharyngeal carcinoma cells. World J Surg Oncol 20:88 (2022). PubMed: 35303867
- Tsaridou S et al. 53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling. EMBO Rep 23:e54483 (2022). PubMed: 35758159
- Cheng H et al. A Novel ATM Antisense Transcript ATM-AS Positively Regulates ATM Expression in Normal and Breast Cancer Cells. Curr Med Sci 42:681-691 (2022). PubMed: 35788947