Anti-Histone H2A antibody [EPR895] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody [EPR895] - BSA and Azide free (AB217838)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Histone H2A 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A antibody [EPR895] - BSA and Azide free (AB217838)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H2A 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody [EPR895] - BSA and Azide free (AB217838)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labeling Histone H2A 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab124781).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Histone H2A antibody [EPR895] - BSA and Azide free (AB217838)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H2A 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

• IP

Lab

Immunoprecipitation - Anti-Histone H2A antibody [EPR895] - BSA and Azide free (AB217838)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

All lanes:

Immunoprecipitation - Anti-Histone H2A antibody [EPR895] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h2ax-antibody-epr895-nuclear-marker-ab124781'>ab124781</a>)

Predicted band size: 15 kDa

true

Exposure time: 20min

• WB

Lab

Western blot - Anti-Histone H2A antibody [EPR895] - BSA and Azide free (AB217838)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

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

Anti-His tag antibody (ab213204) did not detect the 14 kDa protein, indicating that the His tag may have been cleaved from the 14 kDa protein.

All lanes:

Western blot - Anti-Histone H2A antibody [EPR895] - Nuclear Marker (<a href='/en-us/products/primary-antibodies/histone-h2ax-antibody-epr895-nuclear-marker-ab124781'>ab124781</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His tag whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a full-length human Histone H2AX (P16104) expression vector containing a His-tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a full-length human Histone H2A type 1-A (Q96QV6) Expression vector containing a His-tag whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a full-length human Histone H2A type 2-A (Q6FI13) expression vector containing a His-tag whole cell lysate 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: 22 kDa

false

Exposure time: 60s