Anti-Histone H3 antibody - Nuclear Marker

• IP

Unknown

Immunoprecipitation - Anti-Histone H3 antibody - Nuclear Marker (AB18521)

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

All lanes:

Immunoprecipitation - Anti-Histone H3 antibody - Nuclear Marker (ab18521)

Predicted band size: 15 kDa

true

Exposure time: 20min

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 antibody - Nuclear Marker (AB18521)

ab18521 staining Histone H3 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab18521 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 antibody - Nuclear Marker (AB18521)

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.

• WB

AbReview18293****

Western blot - Anti-Histone H3 antibody - Nuclear Marker (AB18521)

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

All lanes:

Western blot - Anti-Histone H3 antibody - Nuclear Marker (ab18521) at 1/250 dilution

Lane 1:

Whole cell lysate prepared from <i>Saccharomyces cerevisiae</i> WT (BY4741) at 500000 Cells

Lane 2:

Whole cell lysate prepared from <i>Saccharomyces cerevisiae</i> delta hht1 HHT2 at 500000 Cells

Lane 3:

Whole cell lysate prepared from <i>Saccharomyces cerevisiae</i> delta hht1 hh2-K9, 14, 18R at 500000 Cells

Secondary

All lanes:

HRP conjugated goat anti-rabbit IgG at 1/10000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

true

Exposure time: 10min

This image was kindly supplied by Dr Tobias Eisenberg by Abreview

• WB

Unknown

Western blot - Anti-Histone H3 antibody - Nuclear Marker (AB18521)

All lanes:

Anti-Histone H3 antibody (ab31602) at 1 µg/mL

Lane 1:

Calf thymus histone lysate at 20 µg

Lane 2:

Calf thymus histone lysate at 20 µg with Human Histone H3 peptide (<a href='/en-us/products/unavailable/human-histone-h3-peptide-ab14949'>ab14949</a>)

Lane 3:

Calf thymus histone lysate at 20 µg with Human Histone H3 (mono methyl K4) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-mono-methyl-k4-peptide-ab1340'>ab1340</a>)

Lane 4:

Calf thymus histone lysate at 20 µg with Human Histone H3 (di methyl K4) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-di-methyl-k4-peptide-ab7768'>ab7768</a>)

Lane 5:

Calf thymus histone lysate at 20 µg with Human Histone H3 (tri methyl K4) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-tri-methyl-k4-peptide-ab1342'>ab1342</a>)

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

• WB

Project

Western blot - Anti-Histone H3 antibody - Nuclear Marker (AB18521)

All lanes:

Western blot - Anti-Histone H3 antibody - Nuclear Marker (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

Predicted band size: 15 kDa

Observed band size: 17 kDa

false