Recombinant Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017)

All lanes : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 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 - ab211017 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab211017 was shown to react with Indoleamine 2, 3-dioxygenase in treated wild-type A549 cells in Western blot with no 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 ab211017 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.

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on dendritic cells of human spleen is observed (PMID: 21328335, PMID: 25271151).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution

Lane 1 : Human ovary cancer lysate at 20 µg
Lane 2 : Human placenta lysate at 20 µg
Lane 3 : Human tonsil lysate at 20 µg
Lane 4 : SK-OV-3 (Human ovarian cancer cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3: 3 minutes; Lane 4: 15 seconds.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 50ng/ml IFN-γ for 16 hours or untreated, labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/2000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The signal increased after treatment with IFN-γ (50 ng/ml) for 16 hours on HeLa cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

All lanes : Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with 50ng/ml Interferon-gamma (IFN-gamma, ab51240) for 16 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The level of Indoleamine 2, 3-dioxygenase expression can be induced by IFN-γ treatment (PMID 16368976).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on dendritic cells of human tonsil is observed (PMID: 21328335, PMID: 25271151).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on endothelial cells of human placenta is observed (PMID: 21328335, PMID: 25271151).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human endometrium cancer tissue labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on human endometrium cancer is observed (PMID: 26155395).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 50ng/ml IFN-? for 16h (red) or untreated (green), labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/500 dilution compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody. 

 

 

Indoleamine 2, 3-dioxygenase was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate treated with 50ng/ml IFN-γ for 16h with ab211017 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab211017 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa treated with 50ng/ml IFN-γ for 16h whole cell lysate 10 µg (Input).

Lane 2: ab211017 IP in HeLa treated with 50ng/ml IFN-γ for 16h whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab211017 in HeLa treated with 50ng/ml IFN-γ for 16h whole cell lysate.

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

Exposure time: 1 second.