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, 5µg of ab31830 (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.

Overlay histogram showing HeLa cells stained with ab31830 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab31830, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

All lanes : Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
Lane 2 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg
Lane 3 : Histone H4 Recombinant Protein at 0.1 µg
Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 14 kDa
Observed band size: 13 kDa why is the actual band size different from the predicted?

All lanes : Anti-Histone H4 antibody [mAbcam 31830] - ChIP Grade (ab31830) at 5 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
Lane 2 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg
Lane 3 : Histone H4 Recombinant Protein at 0.1 µg
Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg
Lane 5 : Histone H2A Recombinant Protein at 0.1 µg
Lane 6 : Histone H2B Recombinant Protein at 0.1 µg
Lane 7 : Histone H1 Recombinant Protein at 0.1 µg
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
Lane 9 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
Lane 10 : Histone H4 Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
Lane 11 : Histone H3.1 Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
Lane 12 : Histone H2A Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
Lane 13 : Histone H2B Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml
Lane 14 : Histone H1 Recombinant Protein at 0.1 µg with Human Histone H4 peptide (ab13843) at 5 µg/ml

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 14 kDa
Observed band size: 13 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

Histone H4 was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to Histone H4 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31830.
Secondary: Protein G-HRP at 1/500 dilution.
Band: 13kDa: Histone H4; non specific - 25 and 55kDa: We are unsure as to the identity of this extra band.

IHC image of Histone H4 staining in human breast fibroadenoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 ab31380, 0.1µg/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.

ab31830 stained in heLa cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab31830 at 5µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) used at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.