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

Alarcon V et al investigates the effects of pargyline due to inhibition of LSD1. Levels of LSD1 protein were reduced using siRNA (siLSD1). Huh7 (Human hepatocellular carcinoma) cells were treated with and without siLSD1 and incubated for 24 hours before transfection of HBV genome (A). Western blot analysis shows the reduction of LSD1 after treatment. Covalent post-translational modifications on Histone H3 was determined by ChIP using ab1220 as one of the specific antibodies (C-G). HBV cccDNA and cytoplasmic levels were determined by qPCR (H).

ab1220 staining Histone H3 (di methyl K9) in HeLa (Human cervix adenocarcinoma epithelial cell) cells. The cells were fixed with 4% PFA (10 mins), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab1220 at 5 µg/ml and ab190573, Rabbit monoclonal to alpha Tubulin (Alexa Fluor^® 647), at 2 µg/ml (shown in red). The secondary antibody (shown in green) was ab150117, Alexa Fluor^® 488 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1h at room temperature. Nuclear DNA was labelled with DAPI (shown in blue).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
This product also gave a positive outcome under the same testing conditions in HeLa cells fixed with 100% methanol (5 mins).

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.
 

Chen L et al investigates role of Trim 28 (Tripartite motif containing 28) in the epithelial to mesenchymal transition which is implicated in cancer metastasis. ChIP was performed using ab1220. ChIP assay was performed in Trim 28 knockdown A549 cells and control (A). ChIP assay was performed in control and Trim28 expressed in A549 cells (B).

ChIP was performed using ab1220. Naïve Mouse ES cells were resuspended in PBS and crosslinked with 1% formaldehyde for 5 minutes at room temperature. Crosslinking was quenched with 125m M glycine. A mouse anti IgG (ab18413) was used as a control. ChIP -seq for H3K9me2 in ES cells treated with and without vitamin C (image a). H3K9me2 signal genome-wide was plotted comparing untreated and vitamin C-treated cells (image b). Fold change for average H3K9me2 signal at gene promoters. Most gene promoters display a reduction in H3K9me2 in vitamin C-treated ES cells (image c). ChIP-qPCR for H3K9me2 in ES cells with or without vitamin C at gene promoters. ChIP for IgG was performed as a negative control (image d). Asterisks represent P < 0.05 by t test

Blocking buffer: 5% milk

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ab1220 staining human kidney sections by IHC-P using EXPOSE IHC detection kit (ab80436). Formalin fixed paraffin embedded tissue sections were pre-treated using heat mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (pH6) for 30 mins. The section was incubated with ab1220, 5µg/ml, for 1 hour at room temperature. DAB was used as the chromogen and the section was counterstained with haematoxylin and mounted with DPX.

ELISA using ab1220 at varying antibody concentrations.

The purple line indicates binding to the Human Histone H3 (di methyl K9) peptide ab1772. Binding to the following peptides was not seen:
Human Histone H3 (unmodified ) peptide (ab2903),
Human Histone H3 (mono methyl K9) peptide (ab1771),
Human Histone H3 (tri methyl K9) peptide (ab1773),
Human Histone H3 (di methyl K27) peptide (ab1781),
Human Histone H3 (tri methyl K27) peptide (ab1782),
Human Histone H3 (mono methyl K4) peptide (ab1340),
Human Histone H3 (di methyl K4) peptide (ab7768),
Human Histone H3 (tri methyl K4) peptide (ab1342).

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

Lane 1 : Marker.

All blocking and antibody incubation steps were done with 5% milk in 20mM Tris-HCL, and 0.1% TWEEN-20.

ab1220 staining Histone H3 (di methyl K9) in Mouse Tissue sections (Liver, P5) by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% BSA for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in PBS/Triton 0,05%) for 3 hours at 25°C. An Alexa Fluor® 488 conjugated Goat anti-mouse (1/250) was used as the secondary antibody.

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