All lanes : Anti-Histone H3 antibody (ab18521) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
Lane 2 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated at 2.5 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

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

IHC image of Histone H3 staining in human testis 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 ab18521, 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.

Histone H3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 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 ab18521.

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

Band: 17kDa; Histone H3

ICC/IF image of ab18521 stained Hek293 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18521, 1æg/ml) overnight at +4øC. The secondary antibody (green)ÿwas 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43æM.

All lanes : Anti-Histone H3 antibody (ab18521) at 1/250 dilution

Lane 1 : Whole cell lysate prepared from Saccharomyces cerevisiae WT (BY4741).
Lane 2 : Whole cell lysate prepared from Saccharomyces cerevisiae delta hht1 HHT2.
Lane 3 : Whole cell lysate prepared from Saccharomyces cerevisiae delta hht1 hh2-K9, 14, 18R.

Lysates/proteins at 500000 cells per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 10 minutes


Blocking performed using 3% BSA for 16 hours at 4°C.Lysates prepared using 5% Trichloroethane.

See Abreview

All lanes : Anti-Histone H3 antibody (ab31602) at 1 µg/ml

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Human Histone H3 peptide (ab14949) at 1 µg/ml
Lane 3 : Calf thymus histone lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 1 µg/ml
Lane 4 : Calf thymus histone lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 1 µg/ml
Lane 5 : Calf thymus histone lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Predicted band size: 15 kDa
Observed band size: 15 kDa



ab18521 recognises histone H3 as well as modified histone H3 at approximately 15 kDa(lane1). This is demonstrated by the blocking using the immunising unmodified histone H3 peptide (lane2), histone H3 mono methyl K4 (lane3), histone H3 di methyl K4 (lane4) and histone H3 tri methyl K4 (lane5).