All lanes : Anti-IL-1 beta antibody [EPR21086] (ab216995) at 1/1000 dilution

Lane 1 : Wild-type THP-1 untreated cell lysate
Lane 2 : Wild-type THP-1 LPS-treated (3 h, 100 ng/ml) cell lysate
Lane 3 : IL1B knockout THP-1 untreated cell lysate
Lane 4 : IL1B knockout THP-1 LPS-treated (3 h, 100 ng/ml) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Observed band size: 27-32 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab216995 observed at 27-32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab216995 was shown to react with IL-1 beta in wild-type THP-1 cells in Western blot with loss of signal observed in IL1B knockout cell line ab273762 (knockout cell lysate ab275508). Wild-type THP-1 and IL1B knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab216995 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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.

Human IL-1 beta concentration was interpolated from the standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human IL-1 beta ELISA kit (ab229384). Wild-type and IL-1 beta knockout THP-1 cells (ab273762) were assessed in duplicate (n=2). Cells were either treated with LPS (100 ng/ml, 3 h) then ATP (5 mM, 45 min) to induce expression of IL-1 beta or not treated. Data are represented as the mean and error bars represent standard deviation.

Allele-1: 47 bp deletion in exon 4.