ChIP - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

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  ab8580 (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.

Peptide Array - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

All batches of ab8580 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - tri methyl K4 peptide (ab1342), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification.

1. ab1340 - Histone H3 - mono methyl K4
2. ab1342 - Histone H3 - tri methyl K4
3. ab1771 - Histone H3 - mono methyl K9
4. ab1772 - Histone H3 - di methyl K9
5. ab1773 - Histone H3 - tri methyl K9
6. ab1780 - Histone H3 - mono methyl K27
7. ab1781 - Histone H3 - di methyl K27
8. ab1782 - Histone H3 - tri methyl K27
9. ab7228 - Histone H3 - unmodified
10. ab7768 - Histone H3 - di methyl K4

Western blot - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

Secondary
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?)


Exposure time : 8 minutes

Western blot - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

All lanes : Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1/5000 dilution

Lane 1 : S. cerevisiae whole cell lysate with WT
Lane 2 : S. cerevisiae whole cell lysate with set1
Lane 3 : S. cerevisiae whole cell lysate with bre2
Lane 4 : S. cerevisiae whole cell lysate with sdc1
Lane 5 : S. cerevisiae whole cell lysate with shg1
Lane 6 : S. cerevisiae whole cell lysate with spp1
Lane 7 : S. cerevisiae whole cell lysate with swd1
Lane 8 : S. cerevisiae whole cell lysate with swd3

Lysates/proteins at 40 µg per lane.


Performed under reducing conditions.

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


Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000. Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr. Blots were washed 6X for 10 min each in PBS with 0.1% Tween-20 before addition of secondary antibodies. Secondary antibodies were diluted 1/2,000 in blocking buffer and incubated with blots for 2 hr at RT. Secondary blots were washed 4X for 10 min each in PBS with 0.1% Tween-20 and 2X for 10 min each in PBS.

John E. Mueller and J. Ruth German (Mary Bryk lab)

Immunocytochemistry - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

ab8580 staining cultured human primary fibroblasts by ICC.  Cells were PFA fixed and permeabilized in TritonX100 and saponin prior to blocking with 1% BSA for 1 hour at RT.  The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C.  A FITC-conjugated rabbit anti-rabbit IgG antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Dr Eva Bartova

Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

Staining (green) with the anti-trimethyl Lysine K4 of Histone H3 antibody (ab8580) shows ringing of regions that appear as nucleoplasmic holes. These represent the positions of splicing factor compartments, which are preferentially associated with active genes and highly acetylated histone H3.   

The antibody, as expected, fails to stain heterochromatin (red). 

Kirk McManus in the lab of Michael Hendzel, University of Alberta

Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

IF of primary cultures of bladder cancer which arise from urothelial cells to determine whether the alterations in chromatin during cancer development would be enhanced by the histone modification antibodies. Both cell types gave very weak staining with the trimethyl-K9-H3 antibody.

Wolfgang Schulz

Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

Mouse zygotes stained with the Tri-Methyl K4 histone H3 antibody
(green) and DNA (blue). This modification is readily detected in the two
pronuclei of the zygote.

This image was kindly supplied as part of the review submitted by Dr Maria Elena Torres Padilla (University of Cambridge, UK).

Immunofluorescence - Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

Human female lymphoblast immunostained with ab8580 (1:100)(yellowish green) specific for histone H3 lysine 4 (H3-K4) trimethylation; the DNA is stained red with propidium iodide (PI).Note the inactive X chromosome (arrow) and pericentromeric heterochromatin are largely devoid of this modification.

Ahmad Khalil and Daniel Driscoll, University of Florida College of Medicine.