Anti-Histone H3 (phospho S28) antibody

• ICC/IF

Characteriser

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (phospho S28) antibody (AB5169)

In situ peptide competition was perfomed on paraformaldehyde-fixed HeLa cells. Four 25μl aliquots were made, to which 7.5μg (1.5μl) of no peptide, H3 unmodified peptide (ab2623), H3 phospho S10 peptide (ab11477) or H3 phospho S28 peptide (ab5499) was added, mixed by vortexing and incubated for 1 hour at room temperature. Cells on glass coverslips were fixed with 4% paraformaldehyde (10min) and gently washed twice with PBS, then permeabilized with 0.5% Triton X-100 in PBS (10min) and gently washed three times with PBS. The cells were immunofluorescently labeled with either the peptide-competed antibody or the control antibody (i.e. no peptide) for 30min at room temperature, washed briefly with PBS containing 0.1% Triton X-100 (1 min) and twice with PBS. The cells were then incubated with an appropriate dilution of a secondary antibody at room temperature for 30min, rinsed as above and mounted using a 90% glycerol in PBS mount

This image was submitted as part of a review by Kirk McManus, University of British Columbia

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (phospho S28) antibody (AB5169)

Indian Muntjac (top panel) and HeLa cells (bottom panel) immunofluorescently labelled with ab5169 (green) at a working dilution of 1/5000. The DNA is counterstained with DAPI and is shown in blue in the top panel and red in the bottom panel. This antibody gives a characteristic staining pattern for Histone H3 (phospho S28) whereby the signal increases in intensity during late G2 and continues to increase until metaphase. Upon entry into anaphase the signal begins to decrease until reaching basal levels by early G1. 100x magnification.

The image was submitted as part of a review by Krik McManus, University of British Columbia

• WB

Lab

Western blot - Anti-Histone H3 (phospho S28) antibody (AB5169)

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 ab5169 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H3 (phospho S28) antibody (ab5169) at 1 µg/mL

All lanes:

Hela Whole Cell Lysate - Colcemid Treated at 2.5 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 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

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Exposure time: 30s

• WB

Collaborator

Western blot - Anti-Histone H3 (phospho S28) antibody (AB5169)

0-4hr old wee shRNA embryos (lane 2) should display elevated phH3Ser28 levels relative to 0-4hr old EGFP shRNA embryos (lane 1). Blocked with 10% BSA.

All lanes:

Western blot - Anti-Histone H3 (phospho S28) antibody (ab5169) at 1/1000 dilution

Lane 1:

Wild type 0-4 hour old fruit fly embryos

Lane 2:

0-4 hour old fruit fly embryos expressing wee RNAi

Secondary

All lanes:

Donkey anti-rabbit IgG, Horseradish Peroxidase-L at 1/10000 dilution

Predicted band size: 15 kDa

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Exposure time: 10min

Image courtesy of Richelle Sopko, Harvard University, U.S.A.

• PepArr

Lab

Peptide Array - Anti-Histone H3 (phospho S28) antibody (AB5169)

All batches of ab5169 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - phospho S28 peptide (ab5499), indicating that this antibody specifically recognises the Histone H3 - phospho S28 modification.

ab17163 - Histone H3 unmodified

ab5499 - Histone H3 - phospho S28

ab11477 - Histone H3 - phospho S10

• WB

CiteAb

Western blot - Anti-Histone H3 (phospho S28) antibody (AB5169)

Histone H3 (phospho S28) western blot using anti-Histone H3 (phospho S28) antibody ab5169. Publication image and figure legend from Pamonsinlapatham, P., Hadj-Slimane, R., et al., 2008, PLoS One, PubMed 18682833.

ab5169 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab5169 please see the product overview.

RG27-induced inhibition of RasGAP-Aurora B interaction and of Aurora B kinase activity.(A,B,C) RasGAP/Aurora B co-capture assay. Upper panels : A RasGAP pulldown assay was performed from HeLa cells (A), HCT116 cells (B) and Panc 10.05 cells (C) expressing control aptamer C6 or RG27, using either a RasGAP-SH2-interacting peptide (DOK) coupled to CNBr-sepharose beads or uncoupled beads (Cont.). Captured RasGAP and Aurora B proteins were revealed by Western blot. Lower panels : Western blot experiments were performed from a fraction of the total HeLa, HCT116, and Panc 10.05 cell lysates used in the pulldown assays, and from lysates of non-transfected cells (NT), using RasGAP and Aurora B specific antibodies. (D) Aurora B kinase activity. The content of phosphorylated Histone H3 was determined by Western blot from soluble protein extracts of HeLa cells expressing either C6 or RG27 peptide aptamers. A Western blot using a γ-tubulin antibody was performed to obtain a loading control. Histogram shows the quantification of three independent experiments.

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