Anti-Histone H2A.X antibody - ChIP Grade

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

ab20669 staining Histone H2A.X in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab20669 at 0.1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

IHC image of Histone H2A.X staining in Human Lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20669, 0.2 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

IHC image of ab20669 staining Histone H2A.X in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20669, 0.5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ChIP

Lab

ChIP - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

Chromatin was prepared from HeLa cells 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 ab20669 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

• IP

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Immunoprecipitation - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

Histone H2A.X was immunoprecipitated using 0.5mg Rat Testis whole tissue extract, 5µg of Rabbit polyclonal to Histone H2A.X and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Rat Testis whole tissue 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab20669.
Secondary : Clean blot (HRP conjugate) at 1/1000 dilution.
Band : 17kDa : Histone H2A.X.

All lanes:

Immunoprecipitation - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

Predicted band size: 15 kDa

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• WB

Lab

Western blot - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

Lanes 1 - 3 : Merged signal (red and green). Green - ab20669 observed at 17 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab20669 was shown to recognize Histone H2A.X in wild-type HAP1 cells as signal was lost at the expected MW in H2AFX (Histone H2A.X) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and H2AFX (Histone H2A.X) knockout samples were subjected to SDS-PAGE. ab20669 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

H2AFX (Histone H2A.X) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK 293 whole cell lysate at 20 µg

Predicted band size: 15 kDa

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• WB

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Western blot - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

All lanes:

Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg

Lane 2:

HeLa Histone Preparation Nuclear Lysate at 2.5 µg

Lane 3:

Histone H2A Recombinant Protein (negative control) at 0.1 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

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Exposure time: 20min

• WB

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Western blot - Anti-Histone H2A.X antibody - ChIP Grade (AB20669)

All lanes:

Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/mL

Lane 1:

Lung (Mouse) Tissue Lysate at 10 µg

Lane 2:

Testis (Mouse) Tissue Lysate at 10 µg

Lane 3:

Lung (Rat) Tissue Lysate at 10 µg

Lane 4:

Testis (Rat) Tissue Lysate - normal tissue (<a href='/en-us/products/unavailable/testis-rat-tissue-lysate-normal-tissue-ab29388'>ab29388</a>) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa,55 kDa

true

Exposure time: 8min