All lanes : Anti-Lin28B antibody [EPR18717] (ab191881) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : LIN28B knockout HEK293T cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : SW480 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: 27 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab191881 observed at 36 kDa. Red - loading control ab7291 observed at 50 kDa.

ab191881 Anti-Lin28B antibody [EPR18717] was shown to specifically react with Lin28B in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab265066 (knockout cell lysate ab257504) was used. Wild-type and Lin28B knockout samples were subjected to SDS-PAGE. ab191881 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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 20000 dilution for 1 hour at room temperature before imaging.

 

Homozygous: 7 bp deletion in exon 2.

Representative images of LIN28B knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T 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.