All lanes : Anti-ATG16L1 antibody [5H9A11] (ab233796) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATG16L1 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 68 kDa

Lanes 1- 4: Merged signal (red and green). Green - ab233796 observed at 68 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

 ab233796 was shown to react with ATG16L1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265263 (knockout cell lysate ab256842) was used. Wild-type HeLa and ATG16L1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab233796 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-ATG16L1 antibody [EPR15638] - N-terminal (ab187671) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATG16L1 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 68 kDa

Lanes 1- 4: Merged signal (red and green). Green - ab187671 observed at 68 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab187671 was shown to react with ATG16L1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265263 (knockout cell lysate ab256842) was used. Wild-type HeLa and ATG16L1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab187671 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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-ATG16L1 antibody [EPR15638] - N-terminal (ab187671) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATG16L1 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Daudi 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: 68 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab187671 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab187671 Anti-ATG16L1 antibody [EPR15638] was shown to specifically react with ATG16L1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265263 (knockout cell lysate ab256842) was used. Wild-type and ATG16L1 knockout samples were subjected to SDS-PAGE. ab187671 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.

Allele-1: 1 bp insertion in exon 1.

 

Allele-2: Insertion of the selection cassette in exon 1.