By peptide array ab14723 has been shown to detect both Histone H2A (phospho S1) and Histone H4 (phospho S1) peptides and not the equivalent unmodified peptides (please see peptide array image on this datasheet). However, by western blot, this antibody shows a preference for Histone H4 (phospho S1) as seen in the western blot images on this datasheet. If you have further questions about the specificity of ab14723, please contact our technical support team, who will be happy to help.
Synthetic peptide corresponding to Histone H4 aa 1-100 (phospho S1) conjugated to keyhole limpet haemocyanin. Also UniProt ID: P04908, Q93077, Q99878 (Histone H2A) Database link: P62805 (Peptide available as ab14724)
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
This antibody was affinity purified using the phospho peptide ab14724, it was then cross-affinity purified using the non-phospho peptide ab14725 in order to remove reactivity with the non-phosphorylated epitope.
Peptide Array analysis of ab14723 at 1:5000 dilution against 40.0 – 0.6 µM peptides: Histone H2A unmodified peptide (Lane 1), Histone H2A (phospho S1) peptide (Lane 2), Histone H4 unmodified peptide (Lane 3) and Histone H4 (phospho S1) peptide (Lane 4). Results show strong binding to the phosphorylated, but not the unmodified versions of these peptides.
Western blot - Anti-Histone H2A (phospho S1) + Histone H4 (phospho S1) antibody (ab14723)
All lanes : Anti-Histone H2A (phospho S1) + Histone H4 (phospho S1) antibody (ab14723) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate - Colcemid Treated at 2.5 µg Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg
Secondary Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (ab201489) at 1/50000 dilution Developed using the ECL technique
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 ab14723 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
Western blot - Anti-Histone H2A (phospho S1) + Histone H4 (phospho S1) antibody (ab14723)This image was submitted as part of a review by Meredith Calvert
All lanes : Anti-Histone H2A (phospho S1) + Histone H4 (phospho S1) antibody (ab14723) at 1/1000 dilution
Lane 1 : YDH8 cka 2-8 ts grown at 30 degrees C Lane 2 : YDH8 cka 2-8 ts grown at 38 degrees C
This image was submitted as part of a review by Meredith Calvert
Cell lysates were prepared from a Casein Kinase 2 temperature sensitive Saccharomyces cerevisiae strain (YDH8 cka2-8ts) grown at either 30°C or 38°C. The kinase is active at °C and inactive at 38°C. Lysates were prepared from asynchronous log-phase cultures and 300µl run on a gel. The Western blot was blocked in milk for 20 minutes and incubated with a 1/1000 dilution of ab14723 for 16 hours. ab14723 detected a band of 15kDa representing Histone H4 (phospho S1) in the lysates grown at 30°C.
IHC image of Histone H2A (phospho S1) + Histone H4 (phospho S1) staining in human breast carcinoma FFPE section, performed on a BondTM 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 ab14723, 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.