Anti-gamma H2A.X (phospho S139) 抗体 [EP854(2)Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells (untreated and treated with H₂O₂) labelling Histone H2A.X (phospho S139) with ab81299 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).

Control 1 : primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunohistochemical staining of paraffin embedded human brain with unpurified ab81299 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling gamma H2A.X with ab81299 at 1/3000 (0.352 ug/ml). Nuclear staining on human cervical carcinoma without lambda protein phosphatase treatment (image A). No signal was detected when tissues were treated with lambda protein phosphatase (image B). The section was incubated with ab81299 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use ab209101. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunofluorescence staining of A549 (Human lung carcinoma cell line) labeling gamma H2A.X (phospho S139) (green) with ab81299.

Cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in PBS-0.2% Triton for 10 minutes. After blocked for 1 hour, primary antibody was diluted in blocking buffer (1/100) and incubated with fixed cells overnight at 4°C. Cells were washed and incubated with secondary antibodies (1/100) for 1 hour at room temperature. All slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was performed using confocal laser scanning microscopy (Lecia) or fluorescence microscopy (Olympus).

Dexamethasone, DEX; Cisplatin, DDP.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling gamma H2A.X with ab81299 at 1/3000 (0.352 ug/ml). Nuclear staining on human breast carcinoma without lambda protein phosphatase treatment (image A). No signal was detected when tissues were treated with lambda protein phosphatase (image B). The section was incubated with ab81299 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use ab209101. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunohistochemical staining of paraffin embedded human brain with purified ab81299 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney transitional cell carcinoma using unpurified ab81299 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

AbReview35291****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

ab81299 staining H2A.X in Human Testis tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in Citric acid. Samples were incubated with primary antibody (1/50 in TBS) for 2 hours at 21°C. A biotin conjugated Anti-Rabbit IgG (goat polyclonal) was used as the secondary antibody at a 1/250 dilution.

This image is courtesy of an Abreview submitted by Carl Hobbs.

• IP

Unknown

Immunoprecipitation - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

ab81299 at 1/40 immunoprecipitating Histone H2A.X (phospho S139) in HepG2 (human hepatocellular carcinoma epithelial) whole cell lysate observed at 15 KDa (lanes 1 and 2).

Lane 1 (input) : HepG2 treated with etoposide and TSA whole cell lysate 10μg
Lane 2 (+) : ab81299 + HepG2 treated with etoposide and TSA whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab81299 in HepG2 treated with etoposide and TSA

For western blotting, ab81299 (Purified) at 1/200 dilution and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

Predicted band size: 15 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Western blot : Rabbit Monoclonal [EP854(2)Y] to gamma H2A.X (phospho S139) ab81299 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 15 kDa in HepG2 Vehicle Control Etoposide (0 uM, 90 min) cell lysates with no signal observed at this size in HepG2 Etoposide treated (100 uM, 90 min) cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc BSA 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/1000 dilution

Lane 1:

HepG2 Vehicle Control Etoposide (0 uM, 90 min) at 20 µg

Lane 2:

HepG2 Etoposide treated (100 uM, 90 min) at 20 µg

Lane 3:

Jurkat Vehicle Control Etoposide (0 uM, 90 min) at 20 µg

Lane 4:

Jurkat Treated Etoposide (100 uM, 90 min) at 20 µg

Lane 5:

Human Brain at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

• WB

Unknown

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Blocking buffer - 5% NFDM/TBST

Diluting buffer - 1% BSA

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/100000 dilution

Lane 1:

HepG2 cell lysate – treated with etoposide at 20 µg

Lane 2:

HepG2 cell lysate – untreated at 20 µg

Secondary

All lanes:

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

Predicted band size: 15 kDa

false

• WB

Unknown

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/500000 dilution

Lane 1:

Jurkat cell lysate - untreated at 10 µg

Lane 2:

Jurkat cell lysate - treated with etoposide at 10 µg

Secondary

All lanes:

HRP Labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

In Western blot, Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (ab124781) staining at 1/2000 dilution.

The identity of the bands between 25kDa and 50kDa are unknown.

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/1000 dilution

Lane 1:

Untreated Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

Jurkat treated with 25µM etoposide for 8 hours whole cell lysate at 20 µg

Lane 3:

Jurkat treated with 25µM etoposide for 8 hours whole cell lysate 20µg, then the membrane treated with Alkaline Phosphatase for 1 hour at 20 µg

Secondary

All lanes:

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

Observed band size: 15 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Mouse anti GAPDH was used as a GAPDH loading control.

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/1000 dilution

Lane 1:

HepG2 Vehicle Control Etoposide (0 uM, 90 min) at 20 µg

Lane 2:

HepG2 Etoposide treated (100 uM, 90 min) at 20 µg

Lane 3:

Jurkat Vehicle Control Etoposide (0 uM, 90 min) at 20 µg

Lane 4:

Jurkat Treated Etoposide (100 uM, 90 min) at 20 µg

Lane 5:

Human Brain at 20 µg

Predicted band size: 15 kDa

false

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

In Western blot, Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (ab124781) staining at 1/2000 dilution.

The identity of the bands between 20kDa and 75kDa are unknown.

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/1000 dilution

Lane 1:

Untreated PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 2:

PC-12 treated with 3µM etoposide for 1-hour whole cell lysate at 20 µg

Lane 3:

PC-12 treated with 3µM etoposide for 1-hour whole cell lysate 20µg, then the membrane treated with Alkaline Phosphatase for 1 hour at 20 µg

Secondary

All lanes:

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

Observed band size: 15 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

In Western blot, Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (ab124781) staining at 1/2000 dilution.

The identity of the bands between 37kDa and 50kDa are unknown.

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 treated with 30µg/ml etoposide for 4 hours, 500ng/ml Trichostatin A(TSA) was then added for additional 4 hours whole cell lysate at 20 µg

Lane 3:

NIH/3T3 treated with 30µg/ml etoposide for 4 hours, 500ng/ml Trichostatin A(TSA) was then added for additional 4 hours whole cell lysate 20µg, then the membrane treated with Alkaline Phosphatase for 1 hour at 20 µg

Secondary

All lanes:

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

Observed band size: 15 kDa

false

Exposure time: 180s

• Dot

Unknown

Dot Blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (AB81299)

Dot blot analysis of Histone H2A.X single phospho peptide pS139 (lane 1) and Histone H2A.X non-phospho peptide (lane 2) with ab81299 at 1/1000. Blocking and diluting buffer was 5% NFDM/TBST. The secondary antibody used was ab97051 Peroxidase conjugated Goat Anti-Rabbit IgG, (H+L) at 1/100,000.