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A sinthetic non-phosphopeptide derived from human Histone H2A.X around the phosphorylation site of serine 140 (Q-A-SP-Q-E)
Our Abpromise guarantee covers the use of ab47503 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use 2 µg for 25 µg of chromatin.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|WB||1/500 - 1/1000. Detects a band of approximately 42 kDa (predicted molecular weight: 15 kDa).|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
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, 2µg of ab47503 (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.
IHC image of ab47503 staining Histone H2A.X 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 ab47503, 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.
ab47503 staining Histone H2A.X in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab47503 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green). AlexaFluor®350 WGA was used at a 1/200 dilution and incubated for 1h with the cells, to label plasma membranes (shown in blue). Nuclear DNA was labelled in red with 1.25 μM DRAQ5™ (ab108410), which was added to the secondary antibody mixture. A secondary only negative control is displayed, which indicates that the Histone H2A.X staining observed is due to primary antibody specificity and not to unspecific binding of the secondary antibody to the cells.
Left image : Untreated
Right image : Treated with PeptideImmunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using ab47503 at a 1/50 dilution.
Left image : Untreated
Right image : Treated with Peptide
ab47503 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"