Histone H3 (acetyl K27) Mouse Monoclonal Antibody [A6D7]

☑ Cell treatment (CT)

Western blot analysis of Histone H3 (acetyl K27) on different lysates with Mouse anti-Histone H3 (acetyl K27) antibody (HA600047) at 1/1,000 dilution.

Lane 1: NIH/3T3 cell lysate
Lane 2: NIH/3T3 treated with 1μM TSA for 18 hours cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 10 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 (HA600047) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of Histone H3 (acetyl K27) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (HA600047, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:20,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: 293 cell lysate
Lane 2: MCF-7 cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: PC-12 cell lysate

☑ Cell treatment (CT)

Western blot analysis of Histone H3 (acetyl K27) on HeLa cell lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (HA600047, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:20,000 dilution was used for 1 hour at room temperature.
Lane 1: Untreated HeLa whole cell lysates
Lane 2: HeLa treated with Trichostatin A whole cell lysates

Immunocytochemistry analysis of HUVEC cells labeling Histone H3 (acetyl K27) with Mouse anti-Histone H3 (acetyl K27) antibody (HA600047) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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 Mouse anti-Histone H3 (acetyl K27) antibody (HA600047) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) 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 (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) was used as the secondary antibody at 1/1,000 dilution.

Immunocytochemistry analysis of NIH/3T3 cells labeling Histone H3 (acetyl K27) with Mouse anti-Histone H3 (acetyl K27) antibody (HA600047) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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 Mouse anti-Histone H3 (acetyl K27) antibody (HA600047) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) 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 (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) was used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Histone H3 (acetyl K27) antibody. 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 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA600047, 1/600) for 30 minutes 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 Histone H3 (acetyl K27) was done on HeLa cells.

The cells were fixed, permeabilized and stained with the primary antibody (HA600047, 1ug/ml) (red) compared with Rabbit IgG, monoclonal - Isotype Control (green). After incubation of the primary antibody at +4℃ for 1 hour, the cells were stained with a Alexa Fluor®488 conjugate-Goat anti-Mouse IgG Secondary antibody at 1/1000 dilution for 30 minutes at +4℃ (dark incubation).Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

☑ Cell treatment (CT)

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with 500ng/mL TSA for 4 hours with Histone H3 (acetyl K27) (HA600047) or Normal Mouse 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.