IHC image of Histone H1x staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab31972, 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.
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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
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 ab31972 (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.
Western blot - Anti-Histone H1x antibody - ChIP Grade (ab31972)
All lanes : Anti-Histone H1x antibody - ChIP Grade (ab31972) at 1 µg/ml
Lane 1 : HeLa nuclear lysate Lane 2 : HeLa nuclear lysate with Human Histone H1x peptide (ab18052) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Predicted band size: 35 kDa Observed band size: 35 kDa Additional bands at: 98 kDa (possible non-specific secondary antibody binding)
ab31972 recognises histone H1.X in HeLa nuclear extract at 35 kDa (lane 1), which is blocked using the immunizing peptide ab18052.
ICC/IF image of ab31972 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab31972, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Izquierdo-Bouldstridge A et al. Histone H1 depletion triggers an interferon response in cancer cells via activation of heterochromatic repeats. Nucleic Acids Res45:11622-11642 (2017).
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Mayor R et al. Genome distribution of replication-independent histone H1 variants shows H1.0 associated with nucleolar domains and H1X associated with RNA polymerase II-enriched regions. J Biol Chem290:7474-91 (2015).
Read more (PubMed: 25645921) »