Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H2A.X with purified ab124781 at a dilution of 1/1000. Cells were fixed with either 4% PFA (top) or 100% methanol (bottom) and permeabilized with 0.1% TritonX-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.

ab7291 mouse anti-Tubulin (1/1000) followed by ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were used to label tubulin. DAPI was used as the nulear counterstain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Histone H2A.X with purified ab124781 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Histone H2A.X with unpurified ab124781 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H2A.X with purified ab124781 at 1/100 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

• WB

Unknown

Western blot - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

All lanes:

Western blot - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (ab124781) at 1/1000 dilution

Lane 1:

Raji lysate at 10 µg

Lane 2:

Fetal kidney lysate at 10 µg

Lane 3:

Human heart lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti Rabbit IgG at 1/2000 dilution

Predicted band size: 15 kDa

Observed band size: 14 kDa

false

• WB

Lab

Western blot - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (ab124781) at 1/3000 dilution

Lane 1:

Raji cell lysate at 20 µg

Lane 2:

HEK293 cell lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Lane 4:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 15 kDa

Observed band size: 14 kDa,22 kDa

false

• WB

Lab

Western blot - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

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] (<a href='/en-us/products/primary-antibodies/gamma-h2ax-phospho-s139-antibody-ep8542y-ab81299'>ab81299</a>) 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='/en-us/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-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

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] (<a href='/en-us/products/primary-antibodies/gamma-h2ax-phospho-s139-antibody-ep8542y-ab81299'>ab81299</a>) 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='/en-us/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-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

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] (<a href='/en-us/products/primary-antibodies/gamma-h2ax-phospho-s139-antibody-ep8542y-ab81299'>ab81299</a>) 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='/en-us/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

• IP

Lab

Immunoprecipitation - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

Histone H2A.X was immunoprecipitated using 5ug of ab124781 from 200ul of HeLa whole cell extract lysate diluted to 0.5mg/ml in RIPA and 50ul of Protein G magnetic beads. No antibody was added to the control (-).

The antibody was incubated under agitation with the Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C. 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab124781 at 1ug/ml. The Secondary antibody was Mouse monoclonal SB62a Anti-Rabbit IgG light chain (HRP) (ab99697) at 1/10,000 dilution.

Band : 15kDa; Histone H2A.X

All lanes:

Immunoprecipitation - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (ab124781)

Predicted band size: 15 kDa

true

Exposure time: 20min

• WB

Lab

Western blot - Anti-Histone H2A.X antibody [EPR895] - Nuclear Marker (AB124781)

~22kDa band may be the monoubiquitinated form of histone H2A

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

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

Lane 1:

Raji cell lysate at 20 µg

Lane 2:

HEK293 cell lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Lane 4:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 15 kDa

Observed band size: 14 kDa,22 kDa

false