Chromatin was prepared from NCCIT cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab254253 (red), and 20 µl of Protein A/G Sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).

Primers and probes are located in the first kb of the transcribed region.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Bmi1 with ab254253 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse colon is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is the ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254253 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human prostatic hyperplasia tissue labeling Bmi1 with ab254253 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human prostatic hyperplasia is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is the ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254253 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling Bmi1 with ab254253 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human gastric cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is the ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab254253 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Bmi1 was immunoprecipitated from 0.35mg of K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab254253 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab254253 at 1/1000 dilution. VeriBlot for IP detection reagent (HRP) (ab131366) was used as secondary antibody at 1/5000 dilution.

Lane 1: K562 whole cell lysate 10 μg (Input). 

Lane 2: ab254253 IP in K562 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254253 in K562 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST

Exposure time: 15 seconds.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cell line labeling Bmi1 with ab254253 at 1/60 dilution (red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling Bmi1 with ab254253 at 1/60 (red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade (ab254253) at 1/5000 dilution

Lane 1 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate
Lane 4 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 5 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 36 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

Exposure time:
Lane 1: 48 seconds
Lanes 2-5: 10 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Molecular weight observed is consistent with what has been described in the literature (PMID: 26110620)

Immunofluorescent analysis of 4% paraformaldehyde, 0.1% triton X-100 fixed, NIH/3T3 (mouse embryonic fibroblast) cells labeling Bmi1 with ab254253 at 1/50 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in NIH/3T3 cells. DAPI was used as a nuclear counterstain (blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) at 1/200 was used as a counterstain.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a AlexaFluor^®488 Goat anti-Rabbit (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized, HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Bmi1 with ab254253 at 1/50 dilution, followed by a AlexaFluor^®488 Goat anti-Rabbit (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in Hela cells. DAPI was used as a nuclear counterstain (blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) at 1/200 was used as a counterstain.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a AlexaFluor^®488 Goat anti-Rabbit (ab150077) secondary antibody at 1/1000 dilution.