Western blot

All lanes : Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895) at 1/500 dilution

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 1 µg/ml
Lane 3 : Calf thymus histone lysate with Histone H3 peptide - di methyl K4 (ab7768) at 1 µg/ml
Lane 4 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 1 µg/ml
Lane 5 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 1 µg/ml
Lane 6 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 1 µg/ml
Lane 7 : Calf thymus histone lysate with Histone H3 peptide - unmodified (ab2903) at 1 µg/ml

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

Performed under reducing conditions.

Predicted band size : 15 kDa
Observed band size : 18 kDa (why is the actual band size different from the predicted?)


Exposure time : 2 minutes

ab8895 is specific for mono-methylated Lysine 4 of histone H3 and does not recognize di- or tri-methyl Lysine 4 nor methylation at Lysine 9. This is shown in lane 2 where the activity of the antibody is specifically blocked by the addition of the immunizing peptide (ab1340).

ChIP - Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab8895 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH and ALDOA (active) and MYO-D (inactive) promoters and over the y-Actin gene (active). Schematic diagram of the y-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.

Electron Microscopy - Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

A: Enlarged region showing the distribution of the H3K4me1 labelling around and within the EC cavities of a murine rod photoreceptor.
B: Schematic representation of the labelling for the H3K4me1 mark. The bar represents 260 nm in A and 1 µm in B.

Image from Kizilyaprak C et al, PLoS One. 2010 Jun 9;5(6):e11039, Fig 6.

Dot Blot - Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

ab8895 used in Dot Blot at a 1/1000 dilution, 18 hours at 4°C.

The dot blot was done using 0.1ug of peptide and screened multiple histone tail modifications to ensure no cross reactivity.

Image courtesy of an anonymous Abreview.

Immunocytochemistry/ Immunofluorescence - Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

Indian muntjac fibroblast cells -  interphase (top left) and prophase (top right and below), stained with:

Mono Methyl K4 antibody, ab8895, (green)
DAPI: red, top left and right; blue, below
Phospho Ser 10 antibody: red (below) and blue (top right)

The perinuclear and perinucleolar heterochromatin domains do not contain Mono Methyl K4.  The Mono Methyl K4, rather, is distributed as small nuclear foci primarily found between DAPI-intense regions of the nucleus. 

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

Flow Cytometry - Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

ab8895 staining mouse embryonic stem cells by flow cytometry. The ES cell colonies were trypsinized and the cells permeabilized before being stained with ab8895 (0.25ug/1.5 x 10⁵ cells). A PE conjugated goat anti-rabbit antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Prof Albrecht Müller