Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y]

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (AB76157)

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.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

Wild-type A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg

Lane 3:

IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg

Lane 4:

IDO1 knockout A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg

Lane 5:

SK-OV-3 cell lysate at 20 µg

Lane 6:

MCF7 cell lysate at 20 µg

Secondary

Lanes 1 - 6:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 6:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 40 kDa,55 kDa

false

• WB

Unknown

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (AB76157)

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

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (ab76157) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) treated with 10ng/ml IFN-gamma for 24 hours whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

false

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR1230Y] (AB76157)

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:

Western blot - 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 at 20 µg

Lane 2:

Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate at 20 µg

Lane 3:

IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate at 20 µg

Lane 4:

IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 42 kDa,56 kDa

false