All lanes : Anti-Histone H3 (methyl K37) antibody [EPR20970] (ab215728) at 1/1000 dilution

Lane 1 : Unmodified recombinant H3 10 ng
Lane 2 : Recombinant mutant H3 chemically modified to mimic H3K37Me, 10ng
Lane 3 : Recombinant mutant H3 chemically modified to mimic H3K37Me2 10 ng
Lane 4 : Recombinant mutant H3 chemically modified to mimic H3K37Me3 10 ng

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 15 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The recombinant proteins were His-tagged.

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Histone H3 (methyl K37) with ab215728 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in NIH/3T3 cell line is observed. DAPI was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counterstain antibody at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

 

 

Flow cytometric analysis of 90% methanol permeabilized, 4% paraformaldehyde-fixed NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Histone H3 (methyl K37) with ab215728 at 1/60 dilution (Red) compared with Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

All lanes : Anti-Histone H3 (methyl K37) antibody [EPR20970] (ab215728) at 1/1000 dilution

Lane 1 : NCCIT (human pluripotent embryonic carcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 15 kDa

Exposure times:
Lane 1: 26 seconds;
Lane 2: 3 minutes;
Lane 3: 81 seconds;
Lane 4: 3 minutes.

Blocking/Dilution buffer: 5% BSA/TBST 

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell line) cells labeling Histone H3 (methyl K37) with ab215728 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HeLa cell line is observed. DAPI was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counterstain antibody at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

 

 

Flow cytometric analysis of 90% methanol permeabilized, 4% paraformaldehyde-fixed HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Histone H3 (methyl K37) with ab215728 at 1/60 dilution (Red) compared with Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

ab215728 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).

Blocking buffer: 5% BSA in TBST.

Primary antibody concentration: 0.1 µg/ml.

Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.