Western blot - Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048)

All lanes : Anti-Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048) at 1/500 dilution

Lane 1 : Histone prep
Lane 2 : Histone prep with Histone H3 peptide - mono methyl K36 (ab1783) at 1 µg/ml
Lane 3 : Histone prep with Histone H3 peptide - di methyl K36 (ab1784) at 1 µg/ml
Lane 4 : Histone prep with Histone H3 peptide - tri methyl K36 (ab1785) at 1 µg/ml
Lane 5 : Histone prep with Histone H3 peptide - unmodified (ab2623) at 1 µg/ml
Lane 6 : Histone prep with Histone H3 peptide - mono methyl K4 (ab1340) at 1 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

ab9048 specifically recognises Histone H3 mono methyl K36. The activity of the antibody is quenched by the addition of the blocking peptide, ab1783 (lane 2).

Immunocytochemistry - Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048)

Staining of interphase nuclei of Hela cells with ab9048 (green) at a working dilution of 1/500. The DNA is stained with DAPI. ab9048 appears to be more associated with heterochromatin (DAPI intense regions) than euchromatin (DAPI less intense regions).

This image was submitted as part of a review by Kirk McManus, University of Columbia

ChIP - Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048)

Chromatin was prepared from U2OS 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, 2 µg of  ab9048 (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). Primers and probes are located in the first kb of the transcribed region.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048)

ab9048 staining Histone H3 (mono methyl K36) in Mouse pancreatic tumor tissue by Immunohistochemistry (Formalin/ PFA-fixed paraffin-embedded tissue sections). The sections were PFA-fixed and subjected to Heat mediated antigen retrieval in citrate and permeabilized with PBST prior to blocking with 5% serum for 1 hour at room temperature. The primary antibody was diluted 1/500 in PBS and incubated with the sample for 8 hours at 4ºC. A Biotin-conjugated Goat anti-Rabbit polyclonal was used as the secondary antibody, diluted 1/1000.

This image is courtesy of an anonymous Abreview.

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048)

ICC/IF image of ab9048 stained HeLa cells. The cells were 4% formaldehyde fixed (10 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 (ab9048, 0.1µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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 1.43µM. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) Hek293, HepG2 and MCF7 cells at 0.1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 0.1µg/ml.

Western blot - Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048)

All lanes : Anti-Histone H3 (mono methyl K36) antibody - ChIP Grade (ab9048) at 1/1400 dilution

Lane 1 : Whole cell lysate prepared from Drosophila BG3 cells
Lane 2 : Whole cell lysate prepared from Drosophila BG3 cells

Lysates/proteins at 500000 cells per lane.

Secondary
HRP donkey anti-rabbit monoclonal at 1/20000 dilution
developed using the ECL technique

Observed band size : 17,42 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 seconds

Image courtesy of an anonymous Abreview.