Western blot - Histone H3 (di methyl K9) antibody [mAbcam 1220] (ab1220)

All lanes : Anti-Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Histone H3 peptide - unmodified at 1 µg/ml
Lane 3 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 1 µg/ml
Lane 4 : Calf thymus histone lysate with Histone H3 peptide - di methyl K9 (ab1772) at 1 µg/ml
Lane 5 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 1 µg/ml
Lane 6 : Calf thymus histone lysate with Histone H3 peptide - di methyl K4 (ab7768) at 1 µg/ml
Lane 7 : Calf thymus histone lysate with Histone H3 peptide - di methyl K27 (ab1781) at 1 µg/ml

Secondary
Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6728) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 17 kDa


Exposure time : 1 minute

ChIP - Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

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  ab1220 (blue), and 20µl 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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first Kb of the transcribed region.
 

Immunocytochemistry/ Immunofluorescence - Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

Interphase HeLa cells were fixed in paraformaldehyde and labelled with ab1220 (1/500). The staining is shown in green. The cells were counterstained with DAPI (red).

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

- Histone H3 (di methyl K9) antibody [mAbcam 1220] (ab1220)

ELISA using ab1220 at varying antibody concentrations.

The purple line indicates binding to the di methyl K9 peptide ab1772. Binding to the following peptides was not seen:
unmodified K9 (ab2903),
mono methyl K9 (ab1771),
tri methyl K9 (ab1773),
di methyl K27 (ab1781),
tri methyl K27 peptide (ab1782),
mono methyl K4 (ab1340),
di methyl K4 (ab7768),
tri methyl K4 (ab1342).

This indicates the specificity of ab1220 for di methyl K9 of Histone H3.

Flow Cytometry - Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

ab1220 staining mouse embryonic stem cells by flow cytometry (gated on all living cells). The cells were trypsinized and stained with the antibody at 1ug/1.5 x 10⁵ cells in a permeabilization buffer. A Cy3 ® conjugated goat anti-mouse antibody was used as the secondary.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (di methyl K9) antibody [mAbcam 1220] - ChIP Grade (ab1220)

ab1220 staining rat liver tissue sections by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in citrate buffer pH 6.0 prior to blocking with 5% serum for 30 minutes at 20°C.  The primary antibody was diluted 1/400 and incubated with the sample for 45 minutes at 20°C.  A HRP-conjugated goat anti-mouse antibody was used as the secondary.

This image is courtesy of an anonymous Abreview