All lanes : Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/10000 dilution

Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : BMI1 knockout MCF7 cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

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.

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.

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).

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

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : BMI1 knockout HEK293T cell lysate
Lane 3 : MOLT-4 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 36 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

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.

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.

Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/20000 dilution (purified) + PC-12 cell lysate at 20 µg

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

Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/20000 dilution (purified)

Lane 1 : K562 cell lysate
Lane 2 : SAOS-2 cell lysate
Lane 3 : SW480 cell lysate
Lane 4 : Molt-4 cell lysate

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/10000 dilution (unpurified)

Lane 1 : K562 cell lysate
Lane 2 : SAOS-2 cell lysate
Lane 3 : SW480 cell lysate
Lane 4 : MOLT4 cell lysate
Lane 5 : HT1080 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 36 kDa

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.

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.

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.

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.

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