All lanes : Anti-TMP21 antibody [EPR9036(B)] (ab134948) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : TMED10 knockout HEK293T cell lysate
Lane 3 : U-87 MG cell lysate
Lane 4 : Raji 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: 25 kDa
Observed band size: 19 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab134948 observed at 19 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab134948 Anti-TMP21 antibody [EPR9036(B)] was shown to specifically react with TMP21 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266227 (knockout cell lysate ab258233) was used. Wild-type and TMP21 knockout samples were subjected to SDS-PAGE. ab134948 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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-TMP21 antibody [EPR9037(B)] (ab133771) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : TMED10 knockout HEK293T cell lysate
Lane 3 : U-87 MG cell lysate
Lane 4 : Raji 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: 25 kDa
Observed band size: 19 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab133771 observed at 19 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab133771 Anti-TMP21 antibody [EPR9037(B)] was shown to specifically react with TMP21 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266227 (knockout cell lysate ab258233) was used. Wild-type and TMP21 knockout samples were subjected to SDS-PAGE. ab133771 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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: 2 bp insertion in exon1