All lanes : Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 2 : Wild-type A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 3 : IP10 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 4 : IP10 knockout A549 IFN-y (ab259377) (100ng/ml, 32h) and TNF-alpha (ab259410) (10ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 5 : THP-1 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 6 : THP-1 IFN-y (ab259377) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 10 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab214668 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab214668 was shown to react with IP10 in wild-type A549 cells in western blot with loss of signal observed in IP10 knockout cell line ab266971 (knockout cell lysate ab256888). Wild-type and IP10 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214668 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^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Allele-1: 4 bp deletion in exon2

 

Allele-2: 1 bp insertion in exon 2.