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Phospho S10 specific clone produced using a synthetic peptide derived from residues 1 - 100 of Human Histone H3, phosphorylated at S10 and tri methylated at K9.
Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising tri methyl K9 and phospho S10 dimodified peptide. Clones were also positively screened against both tri methyl K9 and phospho S10 peptides. Clones were negatively screened against the unmodified version of the peptides. This clone binds to the tri methyl K9 and phospho S10 dimodified peptide and to the phospho S10 peptide, but not to the tri methyl K9 peptide or to equivalent unmodified Histone H3 peptide.
Alternative versions available:
Anti-Histone H3 antibody (phospho S10) (Alexa Fluor® 488) [mAbcam 14955] (ab197502)
Anti-Histone H3 antibody (phospho S10) (Alexa Fluor® 647) [mAbcam 14955] (ab196698)
Our Abpromise guarantee covers the use of ab14955 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration.|
|ChIP||Use at an assay dependent concentration. PubMed: 20864037See Abreview submitted on 5 January 2007.|
|IP||Use at 80 µg/mg of lysate.|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 21734301|
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|WB||Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).|
Indian muntjac and HeLa cells were fixed in paraformaldehyde and labelled with ab14955 (1/2000 dilution) for 30 minutes. The above image contains an interphase and a prophase Indian muntjac cells immunofluorescently labelled with anti-PhosS10 (green) and counterstained with DAPI (red).
IHC image of ab14955 staining Histone H3 (phospho S10) in human kidney 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 ab14955, 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.
Image courtesy of Richelle Sopko, Harvard University, U.S.A
Blocking: 10% BSA
wee shRNA embryos (lane 2) should display elevated phospho H3Ser10 levels relative to wild type
Lane 1 - 8 : Histone H3 (phospho S10) antibody [mAbcam 14955] (ab14955) at 0.5 ug/ml
Lane 1 : Control HeLa Histone prep at 5 ug
Lane 2 : Colcemid treated HeLa Histone prep at 5 ug
Lane 3 : Control HeLa Histone prep at 5 ug with Histone H3 peptide - unmodified (ab7228) at 1 ug/ml
Lane 4 : Colcemid treated HeLa Histone prep at 5 ug with Histone H3 peptide - unmodified (ab7228) at 1 ug/ml
Lane 5 : Control HeLa Histone prep at 5 ug with Histone H3 peptide - phospho S28 (ab14793) at 1 ug/ml
Lane 6 : Colcemid treated HeLa Histone prep at 5 ug with Histone H3 peptide - phospho S28 (ab14793) at 1 ug/ml
Lane 7 : Control HeLa Histone prep at 5 ug with Histone H3 peptide - phospho S10 (ab11477) at 1 ug/ml
By ELISA, ab14955 detects:
the singly modified phospho S10 peptide and the dual modified phospho S10 and tri methyl K9 peptide (the orange and grey lines respectively),
less strongly detects the phospho S28 peptide (purple line),
does not detect the equivalent non-modified Histone H3 peptide for S10 or the singly modified tri methyl K9 peptide (the 2 lines at the bottom of the figure).
The antibody recognises phospho S28 by ELISA (although a phospho S28 peptide does not block the antibody in Western blotting).