Methyl-Histone H3 (Lys27) Antibody Sampler Kit

Western blot analysis of Histone H3 (mono methyl K27) on different lysates with Rabbit anti-Histone H3 (mono methyl K27) antibody (HA722376) at 1/1,000 dilution.

Lane 1: MCF7 cell lysate (20 µg/Lane)
Lane 2: SH-SY5Y cell lysate (20 µg/Lane)
Lane 3: HeLa cell lysate (20 µg/Lane)
Lane 4: A549 cell lysate (20 µg/Lane)
Lane 5: NIH/3T3 cell lysate (20 µg/Lane)
Lane 6: PC-12 cell lysate (20 µg/Lane)

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

Exposure time: 1 minute 50 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 2% BSA/TBST for 1 hour at room temperature. The primary antibody (HA722376) at 1/1,000 dilution was used in 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.

Western blot analysis of Histone H3 (di methyl K27) on different lysates with Rabbit anti-Histone H3 (di methyl K27) antibody (HA722486) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: SH-SY5Y cell lysate (20 µg/Lane)
Lane 3: HCT 116 cell lysate (20 µg/Lane)
Lane 4: C6 cell lysate (20 µg/Lane)
Lane 5: MCF7 cell lysate (20 µg/Lane)
Lane 6: MCF7 treated with 10μM EED226 for 72 hours cell lysate (20 µg/Lane)

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

Exposure time: 30 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 (HA722486) 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.

Western blot analysis of Histone H3 (tri methyl K27) on different lysates with Rabbit anti-Histone H3 (tri methyl K27) antibody (HA722231) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: SH-SY5Y cell lysate (20 µg/Lane)
Lane 3: HCT 116 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: MCF7 cell lysate (20 µg/Lane)
Lane 7: MCF7 treated with 10μM EED226 for 72 hours cell lysate (20 µg/Lane)

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

Exposure time: 25 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 (HA722231) 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.

Western blot analysis of Histone H3 on different lysates with Rabbit anti-Histone H3 antibody (ET1701-64) at 1/20,000 dilution and competitor's antibody at 1/5,000 dilution.

Lane 1: HeLa cell lysate (10 µg/Lane)
Lane 2: A549 cell lysate (10 µg/Lane)
Lane 3: HT-29 cell lysate (10 µg/Lane)
Lane 4: HEK-293 cell lysate (10 µg/Lane)
Lane 5: C2C12 cell lysate (10 µg/Lane)
Lane 6: L-929 cell lysate (10 µg/Lane)
Lane 7: C6 cell lysate (10 µg/Lane)

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

Exposure time: 18 seconds;
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 (ET1701-64) at 1/20,000 dilution and competitor's antibody at 1/5,000 dilution were 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 NIH/3T3 cells labeling Histone H3 (mono methyl K27) with Rabbit anti-Histone H3 (mono methyl K27) antibody (HA722376) at 1/500 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 K27) antibody (HA722376) at 1/500 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.

Immunocytochemistry analysis of MCF7 cells labeling Histone H3 (di methyl K27) with Rabbit anti-Histone H3 (di methyl K27) antibody (HA722486) at 1/100 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 (di methyl K27) antibody (HA722486) at 1/100 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.

Immunocytochemistry analysis of HeLa cells labeling Histone H3 (tri methyl K27) with Rabbit anti-Histone H3 (tri methyl K27) antibody (HA722231) at 1/100 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 (tri methyl K27) antibody (HA722231) at 1/100 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.

Immunofluorescence analysis of paraffin-embedded human liver tissue labeling Histone H3 with Rabbit anti-Histone H3 antibody (ET1701-64) at 1/100 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 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1701-64, green) at 1/100 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Immunohistochemical analysis of paraffin-embedded human colon tissue with Rabbit anti-Histone H3 (tri methyl K27) antibody (HA722231) at 1/1,000 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 (HA722231) at 1/1,000 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 human skin tissue with Rabbit anti-Histone H3 antibody (ET1701-64) at 1/5,000 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 (ET1701-64) at 1/5,000 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.

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells with Histone H3 (mono methyl K27) (HA722376) / Competitor C / 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.

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells with Histone H3 (di methyl K27) (HA722486) 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.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with Histone H3 (tri methyl K27) (HA722231) 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.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with Histone H3 (ET1701-64) / Competitor's antibody / 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.

Histone H3 was immunoprecipitated in 0.2mg HeLa cell lysate with ET1701-64 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1701-64 at 1/20,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: ET1701-64 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of ET1701-64 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 59 seconds

Flow cytometric analysis of MCF7 cells labeling Histone H3 (mono methyl K27).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722376, 1/500) (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).