Lanes 1 - 3: Merged signal (red and green). Green - ab4086 observed at 30 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab4086 was shown to recognize HIST1H1C in wild-type A549 cells as signal was lost at the expected MW in HeLa knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HeLa knockout samples were subjected to SDS-PAGE. Ab4086 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab4086 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 10 min) and incubated with the antibody (ab4086, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

ab4086 Histone H1.2

The antibody was used at a dilution of 1/500

Lane 1: HeLa Histone (5ug) + ab4086 
Lane 2: HeLa Histone (5ug) + ab4086 + 1µg/ml of peptide (Histone H1.2) (ab16936)

Secondary ab: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)

Exposure time: 1 minute
Expected molecular weight: 21.3 kDa

Histone H1.2 - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab4086.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 21kDa; Histone H1.2 - ChIP Grade

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab4086 (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.