Anti-Histone H2A antibody - ChIP Grade

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody - ChIP Grade (AB88770)

IHC image of ab88770 staining Histone H2A 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 ab88770, 0.1μg/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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A antibody - ChIP Grade (AB88770)

IHC image of Histone H2A staining in human breast adenocarcinoma 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 ab88770, 1μg/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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A antibody - ChIP Grade (AB88770)

ICC/IF image of ab88770 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab88770 at 1μg/ml overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti- rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM. This antibody also gave a positive result in formaldehyde fixed (4%) (10min) HeLa, Hek293, HepG2, and MCF-7 cells, and in Hek293 and HepG2 Methanol (100%) (5min) fixed cells.

• ChIP

Lab

ChIP - Anti-Histone H2A antibody - ChIP Grade (AB88770)

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, 5μg of ab88770 (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.

• WB

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Western blot - Anti-Histone H2A antibody - ChIP Grade (AB88770)

All lanes:

Western blot - Anti-Histone H2A antibody - ChIP Grade (ab88770) at 1 µg/mL

Lane 1:

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

Lane 2:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 14 kDa

Observed band size: 100 kDa,17 kDa,36 kDa

true

Exposure time: 30s

• IP

Lab

Immunoprecipitation - Anti-Histone H2A antibody - ChIP Grade (AB88770)

Histone H2A was immunoprecipitated using 0.5mg HeLa whole cell extract, 5μg of Rabbit polyclonal to 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, 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 ab88770.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 14kDa, non specific band - 95kDa : We are unsure as to the identity of this extra band; Histone H2A

All lanes:

Immunoprecipitation - Anti-Histone H2A antibody - ChIP Grade (ab88770)

Predicted band size: 14 kDa

true

Exposure time: 20min

• WB

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Western blot - Anti-Histone H2A antibody - ChIP Grade (AB88770)

All lanes:

Western blot - Anti-Histone H2A antibody - ChIP Grade (ab88770) at 1/1000 dilution

All lanes:

Plasmodium falciparum histone extract at 30000000 Cells

Secondary

All lanes:

HRP-conjugated goat anti-rabbit polyclonal IgG at 1/100000 dilution

Predicted band size: 14 kDa

Observed band size: 16 kDa

true

Exposure time: 1min

This image is courtesy of an anonymous Abreview

• WB

Unknown

Western blot - Anti-Histone H2A antibody - ChIP Grade (AB88770)

All lanes:

Western blot - Anti-Histone H2A antibody - ChIP Grade (ab88770) at 1 µg/mL

Lane 1:

Calf Thymus Histone Preparation Nuclear Lysate at 10 µg

Lane 2:

C.elegans Tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa,17 kDa

true

Exposure time: 2min