重组Anti-Indoleamine 2，3-dioxygenase抗体[EPR1230Y] (ab76157)

All lanes : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (ab76157) at 1/2500 dilution

Lane 1 : Wild-type A549 Treated IFN gamma (25 ng/mL, 48 h) ab281500 cell lysate
Lane 2 : Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate
Lane 3 : IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate
Lane 4 : IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 42-56 kDa why is the actual band size different from the predicted?

Western blot: Anti-IDO1 antibody [EPR1230Y] (ab76157) staining at 1/2500 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab76157 was shown to bind specifically to IDO1. A band was observed at 42/56 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IDO1 knockout cell line. To generate this image, wild-type and IDO1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (ab76157) at 1/2500 dilution

Lane 1 : Wild-type A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate
Lane 2 : Wild-type A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate
Lane 3 : IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate
Lane 4 : IDO1 knockout A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate
Lane 5 : SK-OV-3 cell lysate
Lane 6 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab76157 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab76157 was shown to react with Indoleamine 2, 3-dioxygenase in treated wild-type A549 cells in Western blot with loss of signal observed in treated IDO1 knockout cell line ab266949 (IDO1 knockout cell lysate ab256948). Wild-type A549 and IDO1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab76157 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2500 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.

Lane 1 : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (ab76157) at 1/1000 dilution
Lane 2 : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (ab76157) at 1/1000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 10ng/ml IFN-gamma for 24 hours whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 45 kDa

The expression of Indoleamine 2, 3-dioxygenase can be upregulated by IFN-gamma (PMID: 29685162). We are unsure how to define the extra bands.