All lanes : Anti-NSDHL antibody [EPR14489(2)] (ab199730) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : NSDHL knockout HEK293T cell lysate
Lane 3 : A431 cell lysate
Lane 4 : HeLa 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: 42 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab199730 observed at 38 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab199730 Anti-NSDHL antibody [EPR14489(2)] was shown to specifically react with NSDHL in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266682 (knockout cell lysate ab258082) was used. Wild-type and NSDHL knockout samples were subjected to SDS-PAGE. ab199730 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.

All lanes : Anti-NSDHL antibody [EPR14490] (ab190353) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : NSDHL knockout HEK293T cell lysate
Lane 3 : A431 cell lysate
Lane 4 : HeLa 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: 42 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab190353 observed at 38 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab190353 Anti-NSDHL antibody [EPR14490]  was shown to specifically react with NSDHL in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266682 (knockout cell lysate ab258082) was used. Wild-type and NSDHL knockout samples were subjected to SDS-PAGE. ab190353 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: 1 bp deletion in exon 4

Representative images of NSDHL 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 an EVOS M5000 microscope.