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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
- Flow cytometry [1]
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- Product number
- 702743 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- IDO Recombinant Rabbit Monoclonal Antibody (7H8L17)
- Antibody type
- Monoclonal
- Antigen
- Other
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 7H8L17
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (1), HeLa treated with hIFN-γ (50 ng/mL for 24 hrs) (2), A549 (3), A549 treated with hIFN-γ (50 ng/mL for 24 hrs) (4), SK-OV-3 (5), SK-OV-3 treated with hIFN-γ (50 ng/mL for 24 hrs) (6), THP-1 (7) and THP-1 treated with PMA and hIFN-γ (100 ng/mL PMA, hIFN-γ 100 U/mL for 48 hrs) (8). The blots were probed with Anti-IDO1 Recombinant Rabbit Monoclonal Antibody (Product # 702743,2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036,0.4 µg/mL,1:4000 dilution). A 45 kDa band corresponding to IDO1 that specifically increased upon hIFN-γ treatment was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockout of IDO1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR864924_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of IDO1 was performed by loading 30µg of HeLa wild type (Lane 1), HeLa wild type treated with 50ng/ml IFN gamma for 24hrs (Lane 2), HeLa Cas9 (Lane 3), HeLa Cas9 treated with 50ng/ml IFN gamma for 24hrs (Lane 4), HeLa IDO1 KO (Lane 5) and HeLa IDO1 KO treated with 50ng/ml IFN gamma for 24hrs (Lane 6) whole-cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-IDO Recombinant Rabbit Monoclonal Antibody (7H8L17) (Product # 702743, 2.5µg/ml dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to IDO1. An uncharacterized band was observed in all the samples at ~70kDa except Lane 1.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous IDO1 using Anti-IDO1 Recombinant Rabbit Monoclonal Antibody (Product # 702743, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) is stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Product # R415, 1:300) Panel a-d) shows representative un-treated cells that were stained for detection and localization of IDO1 protein (green) with no signal. Panel e-h) clearly demonstrate cytoplasmic localisation of IDO1 in cells treated with IFN gamma (50 ng/mL 24h). The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockout of IDO was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR864924_LV) and LentiArray Cas9 Lentivirus (Product # A32064). For Flow cytometry analysis, IDO Knock out cells were treated with 50 ng/mL IFN gamma for 24hrs and stained intracellularly using the intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol, with 2 µg/mL IDO Recombinant Rabbit Monoclonal Antibody (7H8L17) (Product # 702743, blue histogram) or with the 2 µg/mL Rabbit IgG Isotype Control (RbNP15), eBioscience™ (14-4616-82, yellow histogram) followed by Goat anti-Rabbit IgG (Heavy chain), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A55053, 1:1000). IFN gamma treated HeLa Cas9 control cells were also stained with the 2 µg/mL IDO Recombinant Rabbit Monoclonal Antibody (7H8L17) (Product # 702743, pink histogram) followed by the secondary antibody. Lossof signal was observed in the IDO KOcells stained with IDO antibody clone 7H8L17 but not in the control Cas9cells. Viable cells were used for analysis, as determined by Fixable Viability Dye eFluor™780 (Product # 65-0865-18).
