Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] - BSA and Azide free

• IP

Supplier Data

Immunoprecipitation - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] - BSA and Azide free (AB277531)

This data was developed using ab277522, the same antibody clone in a different buffer formulation.

Indoleamine 2,3-dioxygenase was immunoprecipitated from 0.35 mg Mouse colon tissue lysate with ab277522 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab277522 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse colon tissue lysate 10μg

Lane 2 : ab277522 IP in mouse colon tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab277522 in mouse colon tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] (<a href='/en-us/products/primary-antibodies/indoleamine-2-3-dioxygenase-antibody-epr24032-22-ab277522'>ab277522</a>)

Predicted band size: 45 kDa

Observed band size: 46 kDa

false

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] - BSA and Azide free (AB277531)

This data was developed using the same antibody clone in a different buffer formulation (ab277522).

Western blot : Anti-IDO1 antibody [EPR24032-22] (ab277522) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab277522 was shown to bind specifically to IDO1. A band was observed at 35/42 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 [EPR24032-22] (<a href='/en-us/products/primary-antibodies/indoleamine-2-3-dioxygenase-antibody-epr24032-22-ab277522'>ab277522</a>) at 1/1000 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: 35 kDa,42 kDa

false

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] - BSA and Azide free (AB277531)

This data was developed using ab277522, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression pattern is consistent with what has been described in the literature (PMID : 30809284; PMID : 19741271).

Exposure time : 26 seconds.

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] (<a href='/en-us/products/primary-antibodies/indoleamine-2-3-dioxygenase-antibody-epr24032-22-ab277522'>ab277522</a>) at 1/1000 dilution

Lane 1:

C2C12 (mouse myoblasts myoblast), whole cell lysate at 20 µg

Lane 2:

C2C12 treated with 50ng/ml IFN-gamma (mouse) for 24 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 45 kDa

Observed band size: 46 kDa

false

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] - BSA and Azide free (AB277531)

This data was developed using ab277522, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression pattern is consistent with what has been described in the literature (PMID : 19741271).

Exposure time : 5.5 seconds.

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] (<a href='/en-us/products/primary-antibodies/indoleamine-2-3-dioxygenase-antibody-epr24032-22-ab277522'>ab277522</a>) at 1/1000 dilution

All lanes:

Mouse colon tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 45 kDa

Observed band size: 46 kDa

false

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] - BSA and Azide free (AB277531)

This data was developed using ab277522, the same antibody clone in a different buffer formulation.

Exposure time : Lane 1-4 : 1 second; Lane 5-8 : 15 seconds; Lane 9-12 : 48 seconds.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) staining at 1/5000 dilution.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Ido1-KO homozygous mice (Strain ID : T011654).

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR24032-22] (<a href='/en-us/products/primary-antibodies/indoleamine-2-3-dioxygenase-antibody-epr24032-22-ab277522'>ab277522</a>) at 1/1000 dilution

Lane 1:

Wild-type mouse epididymis tissue lysate (male case1) at 20 µg

Lane 2:

Wild-type mouse epididymis tissue lysate (male case2) at 20 µg

Lane 3:

Ido1 knockout mouse epididymis tissue lysate (male case1) at 20 µg

Lane 4:

Ido1 knockout mouse epididymis tissue lysate (male case2) at 20 µg

Lane 5:

Wild-type mouse ileum tissue lysate (female case) at 20 µg

Lane 6:

Wild-type mouse ileum tissue lysate (male case) at 20 µg

Lane 7:

Ido1 knockout mouse ileum tissue lysate (male case1) at 20 µg

Lane 8:

Ido1 knockout mouse ileum tissue lysate (male case2) at 20 µg

Lane 9:

Wild-type mouse colon tissue lysate (female case) at 20 µg

Lane 10:

Wild-type mouse colon tissue lysate (male case) at 20 µg

Lane 11:

Ido1 knockout mouse colon tissue lysate (male case1) at 20 µg

Lane 12:

Ido1 knockout mouse colon tissue lysate (male case2) at 20 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

Observed band size: 45 kDa

false