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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
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- Product number
- 710981 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- OTX2 Recombinant Polyclonal Antibody (14HCLC)
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Rat, Mouse, Rabbit and Pig.
- Antibody clone number
- 14HCLC
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-OTX2 Recombinant Polyclonal Antibody (14HCLC) (Product # 710981) and a 32 kDa band corresponding to OTX2 was observed in mouse retina and rat retina as compared to other tissues. Tissue extracts (30 µg lysate) of Mouse retina (Lane 1), Mouse brain (Lane 2), Mouse eye (Lane 3), Mouse lung (Lane 4) and Rat retina (Lane 5) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of NTERA-2 (Lane1) and nuclear enriched extract of NTERA-2 (Lane2). The blots were probed with Anti-OTX2 Recombinant Rabbit Polyclonal Antibody (Product # 710981, 0.5-1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A clear 35 kDa band corresponding to OTX2 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), 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).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence was performed on fixed and permeabilized NTERA-2 cells for detection of OTX2 using Anti-OTX2 Recombinant Rabbit Polyclonal Antibody (Product # 710981, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of OTX2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of OTX2. Panel e) represents control cells with no primary Antibody to assess background.
