All lanes : Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 76 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 - ab199422 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab199422 was shown to react with Annexin-6/ANXA6 in wild-type HeLa cells in western blot with loss of signal observed in ANXA6 knockout cell line ab265677 (ANXA6 knockout cell lysate ab257351). Wild-type and ANXA6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab199422 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat 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: 76 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 - ab201023 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab201023 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with 6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used.  Wild-type and 6/ANXA6 knockout samples were subjected to SDS-PAGE.  ab201023 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-Annexin-6/ANXA6 antibody [EPR19536] (ab201024) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat 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: 76 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 - ab201024 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab201024 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with Annexin-6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used.  Wild-type and Annexin-6/ANXA6 knockout samples were subjected to SDS-PAGE.  ab201024 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: 5 bp deletion in exon 7.

 

Allele-2: 1 bp insertion in exon 7.