All lanes : Anti-160 kD Neurofilament Medium antibody [NF-09] - Neuronal Marker (ab7794) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : NEFM knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 102 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab7794 observed at 150 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab7794 Anti-160 kD Neurofilament Medium antibody [NF-09]  was shown to specifically react with 160 kD Neurofilament Medium in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266741 (knockout cell lysate ab257103) was used. Wild-type and 160 kD Neurofilament Medium knockout samples were subjected to SDS-PAGE. ab7794 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) 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^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Allele-1: 1 bp insertion in exon 2

 

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