Lane 1 : Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543) at 0.5 µg/ml
Lane 2 : Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543) at 1 µg/ml
Lane 3 : Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543) at 2 µg/ml

All lanes : HeLa (treated with Nocodazole) whole cell extract

Secondary
All lanes : Goat Anti-Rat IgG-peroxidase conjugate

Developed using the ECL technique.

Immunocytochemical analysis of HeLa cells fixed and permeabilized with methanol followed by methanol:acetone. Histone H3 (phospho S28) was labeled with ab10543 at 5 μg/mL in ICC/IF (Green). The secondary antobody was a Goat Anti-Rat IgG-FITC conjugate. The nuclear counerstain was DAPI (Blue).

ab10543 staining Histone H3 (phospho S28) in Human neural progenitor cells from iPS cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton and blocked with 1% serum, 0.1% BSA in PBS for 30 minutes at room temperature. Samples were incubated with primary antibody (2ug/ml in blocking buffer) for 16 hours at 4°C. An Alexa Fluor® 488-conjugated Goat anti-rat IgG2a polyclonal was used as the secondary antibody (1/500). Total cells were stained using DAPI (blue)

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All lanes : Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543) at 1 µg/ml

Lane 1 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated
Lane 2 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated with Human Histone H3 (unmodified ) peptide (ab2623) at 0.5 µg/ml
Lane 3 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated with Human Histone H3 (phospho S28) peptide (ab5499) at 0.5 µg/ml
Lane 4 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated with Human Histone H3 (phospho S10) peptide (ab11477) at 0.5 µg/ml

Lysates/proteins at 2.5 µg per lane.

Secondary
All lanes : Peroxidase Conjugated AffiniPure Rabbit Anti-Rat IgG (H+L) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 17 kDa

why is the actual band size different from the predicted?


Exposure time: 20 minutes

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab10543 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

ab10543 staining Histone H3 (phospho S28) in Mouse neural stem cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 4% serum for 1 hour at 20°C. Samples were incubated with primary antibody (1/500 in PBS + 4% serum) for 16 hours at 4°C. An Alexa Fluor® 546-conjugated anti-rat polyclonal was used as the secondary antibody (1/500). DAPI is stainded blue

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ab10543 staining HeLa cells by ICC/IF. The cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X100 in PBS. The cells were then stained with ab10543 at 1/1000 in PBS for 1h at 22°C. A goat anti-rat Alexa Fluro 488 (ab150157) at 1/200 was used as the secondary antibody. Nuclei are stained in red with DAPI. The antibody produces the expected mitotic-associated staining pattern and is extremely strong. MeOH fixed samples were also evaluated and produced a similar, strong staining pattern in mitotic cells.