All lanes : Anti-Bcl6 antibody [EP529Y] (ab33901) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BCL6 knockout HeLa cell lysate
Lane 3 : Ramos cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 79 kDa
Observed band size: 78 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green ab33901 observed at 78 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab33901 Anti-Bcl6 antibody [EP529Y] was shown to react with BCL6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265410 (knockout cell lysate ab257178) was used. Wild-type and Bcl6 knockout samples were subjected to SDS-PAGE. ab33901 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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 10000 dilution for 1 hour at room temperature before imaging.

 

Allele-1: 14 bp deletion in exon 2.

 

Allele-2: Insertion of the selection cassette in exon 2.

 

Representative images of BCL6 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.