Anti-Bmi1 antibody [EPR3745(2)]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarcinoma tissue labelling Bmi1 with unpurifiied ab126783.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunocytochemistry/Immunofluorescence analysis of SW480 cells labelling Bmi1 with unpurified ab126783 at a dilution of 1/100.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labelling Bmi1 with unpurifiied ab126783.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Bmi1 with purified ab126783 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid gland carcinoma tissue labelling Bmi1 with unpurifiied ab126783.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Bmi1 with purified ab126783 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human normal tonsil tissue labelling Bmi1 with unpurifiied ab126783.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

ab126783 (purified) at 1/50 immunoprecipitating Bmi1 in 10 μg K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate (Lanes 1 and 2, observed at 43 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730) instead of ab126783 in K-562 whole cell lysate. For western blotting, ab126783 at 1/500 and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Bmi1 antibody [EPR3745(2)] (ab126783)

Predicted band size: 36 kDa

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• WB

Lab

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/20000 dilution

Lane 1:

K562 cell lysate at 20 µg

Lane 2:

SAOS-2 cell lysate at 20 µg

Lane 3:

SW480 cell lysate at 20 µg

Lane 4:

Molt-4 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 36 kDa

Observed band size: 40 kDa

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• WB

Lab

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Bmi1 knockout HAP1 cell lysate (20 μg)
Lane 3 : U2OS cell lysate (20 μg)
Lane 4 : Molt-4 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab126783 observed at 42 kDa. Red - loading control, ab18058, observed at 37 kDa.

ab126783 was shown to specifically react with Bmi1 when Bmi1 knockout samples were used. Wild-type and Bmi1 knockout samples were subjected to SDS-PAGE. ab126783 and ab18058 (loading control to Vinculin) were both diluted at 1/10 000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed
(ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (ab126783)

Predicted band size: 36 kDa

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• WB

Lab

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Lanes 1- 3 : Merged signal (red and green). Green - ab126783 observed at 37 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab126783 was shown to react with Bmi1 in wild-type MCF7 cells in western blot. Loss of signal was observed when knockout cell line ab262319 (knockout cell lysate ab256851) was used. Wild-type MCF7 and BMI1 knockout MCF7 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab126783 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/10000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

BMI1 knockout MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human BMI1 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-bmi1-knockout-mcf7-cell-line-ab262319'>ab262319</a>)

Lane 3:

A431 cell lysate at 20 µg

Predicted band size: 171 kDa,36 kDa,43 kDa,67 kDa

Observed band size: 175 kDa,37 kDa,67 kDa

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• WB

Lab

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Lanes 1-3 : Merged signal (red and green). Green - ab126783 observed at 37 kDa. Red - loading control ab7291 observed at 50 kDa.

ab126783 Anti-Bmi1 antibody [EPR3745(2)] was shown to specifically react with Bmi1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266514 (knockout cell lysate ab256850) was used. Wild-type and Bmi1 knockout samples were subjected to SDS-PAGE. ab126783 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

BMI1 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human BMI1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-bmi1-knockout-hek-293t-cell-line-ab266514'>ab266514</a>)

Lane 3:

MOLT-4 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 36 kDa

Observed band size: 37 kDa

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• WB

Unknown

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

All lanes:

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/10000 dilution

Lane 1:

K562 cell lysate at 10 µg

Lane 2:

SAOS-2 cell lysate at 10 µg

Lane 3:

SW480 cell lysate at 10 µg

Lane 4:

MOLT4 cell lysate at 10 µg

Lane 5:

HT1080 cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 36 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

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 5 µg of ab126783 [EPR3745(2)]. 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.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

CUT&RUN was performed using ChiC/CUT&RUN pAG-MNAse ab285373, 2.5 x 10^5 NCCIT cells and 5 μg ab126783 [EPR3745(2)]. The resulting DNA was sequenced on the ILLUMNIA NovaSeq 6000 in a depth of 10 million reads. Additional screenshots and mapped roads can be dowanloaded here.

• WB

Lab

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/20000 dilution

All lanes:

PC-12 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 36 kDa,37 kDa

Observed band size: 36 kDa,40 kDa

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• WB

CiteAb

Western blot - Anti-Bmi1 antibody [EPR3745(2)] (AB126783)

Bmi1 western blot using anti-Bmi1 antibody [EPR3745(2)] ab126783. Publication image and figure legend from Shi, M., An, G., et al., 2023, Cancers (Basel), PubMed 37173968.

ab126783 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab126783 please see the product overview.

Correlation between UVRAG expression and therapeutic resistance. (A) Relative expression of UVRAG in HCT8, HCT8-5Fu, and HCT8-60Gy cells. (B) The sensitivity of HCT8-CON and HCT8-UVRAG cells to radiotherapy. (C) UVRAG knockdown increased 5-Fu sensitivity. (D) UVRAG overexpression decreased 5-Fu sensitivity. (E) Relative mRNA expression of UVRAG in HCT116 and HCT116-self cells. (F) Relative mRNA expression of UVRAG in HCT116 DCLK- and HCT116 DCLK+ cells. (G) Western blot of PD-L1, UVARG, ABCG2, BMI1, and SP1 after upregulating UVARG or downregulating UVRAG. The uncropped blots are shown in Figure S1. (H) Spheroid formation in UVRAG overexpressed HCT116 and its control cells. (I) Stem cell score evaluated via ssGSEA in patients with UVRAG higher expression and UVARG lower expression. Independent-samples t-test (A,E,F). Mann Whitney U test (I). * p < 0.05, ** p < 0.01.

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