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?

This data was developed using the same antibody clone in a different buffer formulation (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.

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.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).

All lanes : Anti-Bmi1 antibody [EPR3745(2)] (HRP) (ab197620) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : COMMD3-BMI1 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 36 kDa

ab197620 was shown to recognize Bmi1 in wild-type HAP1 cells as signal was lost at the expected MW in COMMD3-BMI1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and COMMD3-BMI1 knockout samples were subjected to SDS-PAGE. Ab197620 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab197620).

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?

This data was developed using the same antibody clone in a different buffer formulation (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.

ab126783 (purified) at 1/500 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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).

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

 

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

 

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

 

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

 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).

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