Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Intracellular 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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Intracellular 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• ChIP

Lab

ChIP - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

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

• ChIP-seq

Lab

ChIP-sequencing - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Chromatin was prepared from NCCIT cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab254253 [EPR22604-160]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• ChIP-seq

Lab

ChIP-sequencing - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Chromatin was prepared from NCCIT cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab254253 [EPR22604-160]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• IP

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Immunoprecipitation - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

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 lysate 10 μg (Input).

Lane 2 : ab254253 IP in K562 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

All lanes:

Immunoprecipitation - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade (<a href='/en-us/products/primary-antibodies/bmi1-antibody-epr22604-160-chip-grade-ab254253'>ab254253</a>)

Predicted band size: 36 kDa

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• ChIP-seq

Lab

ChIP-sequencing - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Chromatin was prepared from NCCIT cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab254253 [EPR22604-160]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• ChIP-seq

Lab

ChIP-sequencing - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Chromatin was prepared from NCCIT cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 NCCIT cells and 8 µg of ab254253 [EPR22604-160]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

Immunofluorescent analysis of 4% paraformaldehyde, 0.1% tritonX-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.

Control : Secondary antibody only

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Bmi1 antibody [EPR22604-160] - ChIP Grade - BSA and Azide free (AB254475)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 NCCIT (Human pluripotent embryonic carcinoma cell line) cells and 5µg of ab254253 [EPR22604-160]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254253).