WB
Western blot analysis of Histone H2A (acetyl K9) on different lysates with Rabbit anti-Histone H2A (acetyl K9) antibody at 1/1000 dilution.
Lane 1: HeLa whole cell lysate (20 µg/Lane),
Lane 2: HeLa treated with 500 ng/mL TSA for 4 hours whole cell lysate (20 µg/Lane),
Exposure time: 3 minutes;
15% 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 at 1/1000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/300000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells labeling Histone H2A (acetyl K9) with Rabbit anti-Histone H2A (acetyl K9) antibody at 1/50 dilution.
Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Histone H2A (acetyl K9) antibody at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) 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.
IHC
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Histone H2A (acetyl K9) antibody at 1/4000 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 at 1/4000 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.A
IHC
Immunohistochemical analysis of paraffin-embedded mouse skin tissue with Rabbit anti-Histone H2A (acetyl K9) antibody at 1/4000 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 at 1/4000 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.
IHC
Immunohistochemical analysis of paraffin-embedded rat skin tissue with Rabbit anti-Histone H2A (acetyl K9) antibody at 1/4000 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 at 1/4000 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.
ChIP
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with 500ng/mL TSA for 4 hours with Histone H2A (acetyl K9) 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.