All lanes : Anti-IL-8 antibody [EPR22994-255] (ab235584) at 1/1000 dilution

Lane 1 : Wild-type PC-3 Brefeldin A (ab120299)-treated (5 μg/ml, 5 h) cell lysate
Lane 2 : Wild-type PC-3 LPS-treated (2 μg/ml, 6 h) with Brefeldin A (ab120299) (5 μg/ml, 5 h) cell lysate
Lane 3 : CXCL8 knockout PC-3 Brefeldin A (ab120299)-treated (5 μg/ml, 5 h) cell lysate
Lane 4 : CXCL8 knockout PC-3 LPS-treated (2 μg/ml, 6 h) with Brefeldin A (ab120299) (5 μg/ml, 5 h) cell lysate
Lane 5 : A431 cell lysate
Lane 6 : HCT116 cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

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

Lanes 1 - 6: Merged signal (red and green). Green - ab235584 observed at 10 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab235584 was shown to react with IL-8 in wild-type PC-3 cells in Western blot with loss of signal observed in CXCL8 knockout cell line ab273743 (knockout cell lysate ab275520). Wild-type PC-3 and CXCL8 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab235584 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 h at room temperature before imaging.

Allele-1: 11bp deletion in exon 2.

Allele-2: 7bp deletion in exon 2.

Allele-3: 2bp deletion in exon 2.