Histone H3 (mono methyl K9) Recombinant Rabbit Monoclonal Antibody [JE43-27]

Western blot analysis of Histone H3 (mono methyl K9) on different lysates with Rabbit anti-Histone H3 (mono methyl K9) antibody (HA722350) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: U-2 OS cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: C6 cell lysate
Lane 5: COS-1 cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 2 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722350) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of U-2 OS cells labeling Histone H3 (mono methyl K9) with Rabbit anti-Histone H3 (mono methyl K9) antibody (HA722350) at 1/3,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Histone H3 (mono methyl K9) antibody (HA722350) at 1/3,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-Histone H3 (mono methyl K9) antibody (HA722350) at 1/50,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722350) at 1/50,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-Histone H3 (mono methyl K9) antibody (HA722350) at 1/50,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722350) at 1/50,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Flow cytometric analysis of U-2 OS cells labeling Histone H3 (mono methyl K9).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722350, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with Histone H3 (mono methyl K9) (HA722350) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Dot blot analysis of Histone H3 (mono methyl K9) on different proteins with Rabbit anti-Histone H3 (mono methyl K9) antibody (HA722350) at 1/1,000 dilution. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution for 1 hour at room temperature.

Lane 1: Unmodified Histone H3 (negative)
Lane 2: Mono-Methyl-Histone H3 (Lys9) (positive)
Lane 3: Di-Methyl-Histone H3 (Lys9) (negative)
Lane 4: Tri-Methyl-Histone H3 (Lys9) (negative)
Lane 5: Acetyl-Histone H3 (Lys9) (negative)

Proteins loading: 100ng, 50ng, 25ng;

Blocking and dilution buffer: 5% NFDM/TBST;

Exposure time: 6 seconds; ECL: K1801.