Anti-Histone H1.2抗体- ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

ICC/IF image of ab4086 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 10 min) and incubated with the antibody (ab4086, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

IHC image of Histone H1.2 staining in human breast carcinoma FFPE section, performed on a Bond^TM 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 ab4086, 1µg/ml, for 8 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.

• IP

Lab

Immunoprecipitation - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

Histone H1.2 - ChIP Grade 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 ab4086.

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

Band : 21kDa; Histone H1.2 - ChIP Grade

All lanes:

Immunoprecipitation - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

Predicted band size: 21 kDa

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Exposure time: 8min

• ChIP

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ChIP - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab4086 (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

Lab

Western blot - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

Lanes 1 - 3 : Merged signal (red and green). Green - ab4086 observed at 30 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab4086 was shown to recognize HIST1H1C in wild-type A549 cells as signal was lost at the expected MW in HeLa knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HeLa knockout samples were subjected to SDS-PAGE. ab4086 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution 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 H1.2 antibody - ChIP Grade (ab4086) at 1/500 dilution

Lane 1:

Wild-type A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg/mL

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg/mL

Lane 2:

Western blot - Human HIST1H1C (Histone H1.2) knockout A549 cell line (<a href='/products/cell-lines/human-hist1h1c-histone-h12-knockout-a549-cell-line-ab261873'>ab261873</a>)

Lane 3:

HIST1H1C knockout A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 21 kDa

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• WB

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Western blot - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

ab4086 Histone H1.2

The antibody was used at a dilution of 1/500

Lane 1 : HeLa Histone (5ug) + ab4086
Lane 2 : HeLa Histone (5ug) + ab4086 + 1μg/ml of peptide (Histone H1.2) (ab16936)

Secondary ab : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

Exposure time : 1 minute
Expected molecular weight : 21.3 kDa

ab4086 Histone H1.2

The antibody was used at a dilution of 1/500

Lane 1 : HeLa Histone (5ug) + ab4086
Lane 2 : HeLa Histone (5ug) + ab4086 + 1µg/ml of peptide (Histone H1.2) (ab16936)

Secondary ab : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

Exposure time : 1 minute
Expected molecular weight : 21.3 kDa

All lanes:

Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086)

Predicted band size: 21 kDa

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• WB

Lab

Western blot - Anti-Histone H1.2 antibody - ChIP Grade (AB4086)

False colour image of Western blot : Anti-Histone H1.2 antibody - ChIP Grade staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab4086 was shown to bind specifically to Histone H1.2. A band was observed at 37 kDa in wild-type HeLa cell lysates with no signal observed at this size in HIST1H1C knockout cell line ab261794 (knockout cell lysate ab257218). To generate this image, wild-type and HIST1H1C knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk 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 IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Histone H1.2 antibody - ChIP Grade (ab4086) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HIST1H1C knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human HIST1H1C (Histone H1.2) knockout HeLa cell line (<a href='/products/cell-lines/human-hist1h1c-histone-h12-knockout-hela-cell-line-ab261794'>ab261794</a>)

Predicted band size: 21 kDa

Observed band size: 37 kDa

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