Anti-ATM antibody [EPR20100] - ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATM antibody [EPR20100] - ChIP Grade (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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

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 Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

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.

• ChIP

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ChIP - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

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).

• IP

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Immunoprecipitation - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

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 Reaction (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

All lanes:

Immunoprecipitation - Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022)

Predicted band size: 351 kDa

Observed band size: 350 kDa

false

• WB

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Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) at 1/5000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 351 kDa

Observed band size: 351 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

False 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.

All lanes:

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

ATM knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human ATM knockout A549 cell line (<a href='/en-us/products/cell-lines/human-atm-knockout-a549-cell-line-ab276095'>ab276095</a>)

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

U-2 OS cell lysate at 20 µg

Predicted band size: 351 kDa

Observed band size: 350 kDa

false

• WB

Lab

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (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

All lanes:

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

ATM knockout MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human ATM heterozygous knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-atm-heterozygous-knockout-mcf7-cell-line-ab282630'>ab282630</a>)

Lane 3:

Wild-type A549 cell lysate at 20 µg

Lane 4:

ATM knockout A549 ab283811 cell lysate at 20 µg

Predicted band size: 351 kDa

Observed band size: 350 kDa

false

• WB

Supplier Data

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (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

All lanes:

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (ab201022) at 1/1000 dilution

Lane 1:

Human testis lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 4:

293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 5:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 351 kDa

Observed band size: 351 kDa

false

• WB

Supplier Data

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The identity of the lower MW band at approximately 190 kDa is unknown.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

In Western blot, Anti-ATM antibody [EPR20100] - (ab201022) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ATM (phospho S1987) antibody [EPR28058-71] (<a href='/en-us/products/primary-antibodies/atm-phospho-s1987-antibody-epr28058-71-ab315019'>ab315019</a>) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

NIH/3T3 treated with UV-C for 100J/cm2, then recovery 2 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

NIH/3T3 treated with UV-C for 100J/cm2, then recovery 2 hours, whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 349 kDa,124 kDa

false

Exposure time: 81s

• WB

Supplier Data

Western blot - Anti-ATM antibody [EPR20100] - ChIP Grade (AB201022)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The identity of the lower MW band at approximately 190 kDa is unknown.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

In Western blot, Anti-ATM antibody [EPR20100] - (ab201022) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ATM (phospho S1987) antibody [EPR28058-71] (<a href='/en-us/products/primary-antibodies/atm-phospho-s1987-antibody-epr28058-71-ab315019'>ab315019</a>) at 1/1000 dilution

Lane 1:

Untreated MEF (mouse embryo fibroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

MEF treated with UV-C for 100J/m2, then recovery 4 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

MEF treated with UV-C for 100J/m2, then recovery 4 hours, whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 349 kDa,124 kDa

false

Exposure time: 81s