Recombinant Anti-ATM antibody [EPR20100] - BSA and Azide free (ab223533)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR20100] to ATM - BSA and Azide free
- Suitable for: Flow Cyt (Intra), ChIP, IP, ICC/IF, WB
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-ATM antibody [EPR20100] - BSA and Azide free
See all ATM primary antibodies -
Description
Rabbit monoclonal [EPR20100] to ATM - BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), ChIP, IP, ICC/IF, WBmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Human testis lysate; HeLa, PC-12, RAW 264.7, 293 and SH-SY5Y whole cell lysates. ICC/IF: SH-SY5Y and HeLa cells. Flow Cyt (intra): HEK-293 cells. IP: HEK-293 whole cell lysate. ChIP: Chromatin prepared from HCT 116 cells treated with 1mM Hydroxyurea for 16h.
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General notes
ab223533 is the carrier-free version of ab201022.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.2
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR20100 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab223533 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) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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ChIP |
Use at an assay dependent concentration.
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IP |
Use at an assay dependent concentration.
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ICC/IF |
Use at an assay dependent concentration.
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WB |
Use at an assay dependent concentration. Detects a band of approximately 351 kDa (predicted molecular weight: 351 kDa).
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Notes |
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Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
ChIP
Use at an assay dependent concentration. |
IP
Use at an assay dependent concentration. |
ICC/IF
Use at an assay dependent concentration. |
WB
Use at an assay dependent concentration. Detects a band of approximately 351 kDa (predicted molecular weight: 351 kDa). |
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
- Entrez Gene: 11920 Mouse
- Entrez Gene: 300711 Rat
- Omim: 607585 Human
- SwissProt: Q13315 Human
- SwissProt: Q62388 Mouse
- Unigene: 367437 Human
- Unigene: 5088 Mouse
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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 [EPR20100] - ChIP Grade (ab201022) at 1/1000 dilution
Lane 1 : Human testis lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 4 : 293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 5 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 351 kDa
Observed band size: 351 kDaThis data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1 and 2: 3 minutes; Lane 3: 30 seconds; Lane 4: 5 seconds; Lane 5: 1 second
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Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) at 1/5000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 351 kDa
Observed band size: 351 kDa
Exposure time: 10 secondsThis data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
Blocking/Dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) 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: 351 kDaFalse colour image of Western blot: Anti-ATM antibody [EPR20100] - ChIP Grade 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, ab201022 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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All lanes : Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) 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: 351 kDa
Observed band size: 350 kDa why is the actual band size different from the predicted?This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
Anti-ATM antibody [EPR20100] (ab201022) 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, ab201022 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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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ATM with ab201022 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on HeLa cell line.
The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
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Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293 (Human epithelial cell line from embryonic kidney) cells labeling ATM with ab201022 at 1/800 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
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ATM was immunoprecipitated from 0.35 mg of HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate with ab201022 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab201022 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HEK-293 whole cell lysate, 10 μg (Input).
Lane 2: ab201022 IP in HEK-293 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab201022 in HEK-293 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.
ATM cleavage has been documented previously and the fragment pattern is consistent with what has been described in the literature PMID:16849690
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
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Chromatin was prepared from HCT 116 (Human colorectal carcinoma cell line) cells treated with 1mM Hydroxyurea for 16h and non-treated according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab201022 (blue), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab201022).
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This ICC data was generated using the same anti-ATM antibody clone [EPR20100] in a different buffer formulation (cat# ab201022).
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling ATM with ab201022 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on SH-SY5Y cell line.
The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (0)
ab223533 has not yet been referenced specifically in any publications.