Western blot - Anti-Histone H1x antibody (ab17729)
All lanes : Anti-Histone H1x antibody (ab17729) at 0.5 µg/ml
Lane 1 : HeLa whole cell extract Lane 2 : HeLa whole cell extract with Human Histone H1x peptide (ab18053) at 1 µg/ml Lane 3 : HeLa nuclear extract
Predicted band size : 35 kDa Observed band size : 35 kDa ab17729 recognises histone H1.X in HeLa whole cell (lane1) and nuclear extracts (lane3) at 35 kDa, which is efficiently blocked using the immunizing peptide ab18053 (lane2). ab17729 recognises bands at 20 kDa, which is attributed to minor cross-reactivity and may be improved using different blocking conditions.
ICC/IF image of ab17729 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab17729, 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).
ab17729 staining histone H1.X in male cerebellum, showing a distinct and strong nuclear staining pattern at cells in granular and molecular layers. Paraffin embedded human skin tissue was incubated with ab17729 (1/750 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
ab17729 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org
Crawford NP et al. The metastasis efficiency modifier ribosomal RNA processing 1 homolog B (RRP1B) is a chromatin-associated factor. J Biol Chem284:28660-73 (2009).
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