Myc affects the incorporation levels of histone variant H2A.Z.

qChIP was performed using specific antibodies recognizing A. H2A.Z, B. H2A.Zac, Panel D. H2A, E. H2AK5ac and G. H1. All the qChIP values are expressed as % of input and normalized for total histone H3, with the exception of C and F, where H2A.Z acetylation is noramlized for H2A.Z density, and H2AK5 acetylation is normalized for H2A density, respectively. The box plots show the fold change distribution of each acetylated residue for the two subpopulations.

All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
Lane 3 : Histone H2A Recombinant Protein at 0.1 µg
Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg
Lane 5 : Histone H4 Recombinant Protein at 0.1 µg

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 14 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

DENV C colocalizes with histones in Huh7 liver cells.

DEN2 C colocalized with H2A (A), H2B (B), H3 (C) and H4 (D) in Huh7 cells. Cells were transfected with GFP-DEN2 C and fixed in 4% paraformaldehyde 48 h post-transfection. Cells were stained with antibodies against histones and a TRITC secondary antibody. Cells were counterstained with DAPI to visualize the nucleus. GFP-DEN2 C expression is green, histone staining is red and DAPI is blue.

ICC/IF image of ab18255 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 100% methanol (5 min) then permeabilized using 0.1% PBS-Triton and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18255 at 1 µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor^® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

Histone H2A - ChIP Grade was immunoprecipitated using 0.5 mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5 µg of Rabbit polyclonal to and 50 µL of protein G magnetic beads (+). No antibody was added to the control (-).

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

Proteins were eluted by addition of 40 µL SDS loading buffer and incubated for 10 min at 70°C; 10 µL of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18255.

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

Band: 14kDa, non specific bands - 42kDa: We are unsure as to the identity of this extra band; Histone H2A - ChIP Grade

Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 6 µL of ab18255 (blue), and 20 µL 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.

DENV expression disrupts histone oligomerization.

Panel A and B shown:

Huh7 cells were transfected with DEN2 C and/or infected with DEN2 24 h post-transfection. Cells were lysed 24 h post-infection (48 h post-transfection) and lysates were run on 4–12% SDS-PAGE gel. Gels were used in a Western blotting assay with antibodies against histones H2A ab18255 (A), H2B (B), H3 (C) and H4 (D); monomers, dimers and octamers are indicated. Gels were stripped and reprobed with an antibody against actin as a protein loading control. The same amount of protein was loaded in each lane for each gel as a control for expression.

All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) lysate at 20 µg
Lane 2 : HeLa nuclear lysate at 20 µg
Lane 3 : Calf thymus histone lysate at 20 µg
Lane 4 : HeLa lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml
Lane 5 : HeLa nuclear lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml
Lane 6 : Calf thymus histone lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml

Predicted band size: 14 kDa
Observed band size: 14 kDa
Additional bands at: 22 kDa (possible cross reactivity)

ab18255 is partially blocked by the immunizing peptide ab19751. There is an additional band at 22kDa in HeLa lysate which is attributed to cross-reactivity.

All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1/1000 dilution

Lane 1 : Native recombinant octamers K562 cells
Lane 2 : Recombinant Human octamers containing H2A
Lane 3 : Recombinant Human octamers containing H2A.Z.2.1
Lane 4 : Recombinant Human octamers containing H2A.Z.1

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : HRP-conjugated donkey anti-rabbit IgG polyclonal at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 14 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 5 minutes

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