All lanes : Anti-Bmi1 antibody [BMI1/2823] (ab269678)

Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : BMI1 knockout MCF7 cell lysate
Lane 3 : A431 cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 36 kDa

Lanes 1- 4: Merged signal (red and green). Green - ab269678 observed at 36 kDa. Red - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) observed at 50 kDa.

 ab269678 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. ab269678 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) overnight at 4°C at a 2 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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.

Allel-2: 1 bp insertion in exon 2.

Allele-1: 1 bp deletion in exon2

 

Allele-2: 1 bp insertion in exon 2.