Polycomb Group Antibody Sampler Kit

Western blot analysis of KMT6/EZH2 on different lysates with Rabbit anti-KMT6/EZH2 antibody (HA722095) at 1/1,000 dilution.

Lane 1: HEK-293 cell lysate
Lane 2: 293T cell lysate
Lane 3: MCF7 cell lysate
Lane 4: HeLa cell lysate
Lane 5: COS-1 cell lysate
Lane 6: NIH/3T3 cell lysate
Lane 7: PC-12 cell lysate
Lane 8: C6 cell lysate

Lysates/proteins at 20 µg/Lane.
Predicted band size: 85 kDa
Observed band size: 85 kDa

Exposure time: 3 minutes;
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 (HA722095) 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 HeLa cells labeling KMT6/EZH2 with Rabbit anti-KMT6/EZH2 antibody (HA722095) 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-KMT6/EZH2 antibody (HA722095) 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.

Western blot analysis of RING1 on different lysates with Rabbit anti-RING1 antibody (HA721461) at 1/1,000 dilution.

Lane 1: THP-1 cell lysate
Lane 2: Jurkat cell lysate
Lane 3: 22RV1 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 42 kDa
Observed band size: 50 kDa

Exposure time: 1 minute;

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 (HA721461) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

Immunohistochemical analysis of paraffin-embedded human prostate tissue with Rabbit anti-RING1 antibody (HA721461) at 1/200 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 (HA721461) at 1/200 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.

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

Lysates/proteins at 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.

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.

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.

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.