All lanes : Anti-LRP5 antibody [EPR22477-218] (ab223203) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : LRP5 knockout HEK293T cell lysate
Lane 3 : SW620 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: 179 kDa
Observed band size: 180-200 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab223203 observed at 180-200 kDa. Red - loading control ab7291 observed at 50 kDa.

ab223203 Anti-LRP5 antibody [EPR22477-218] was shown to specifically react with LRP5 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266618 (knockout cell lysate ab257202) was used. Wild-type and LRP5 knockout samples were subjected to SDS-PAGE. ab223203 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 500 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.

 

 

Allele-1: 5 bp deletion in exon 2

 

Allele-2: 1 bp insertion in exon 2.

 

Representative images of LRP5 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.