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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Chromatin Immunoprecipitation [1]
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Validation data
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- Product number
- 711585 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- FOXN3 Recombinant Polyclonal Antibody (1HCLC)
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Hors and Cat
- Antibody clone number
- 1HCLC
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on Nuclear enriched extracts (30 µg lysate) of Jurkat (Lane 1), Hep G2 (Lane 2), KARPAS 299 (Lane 3), HT-29 (Lane 4), THP-1 (Lane 5), U-937 (Lane 6) and U-2 OS (Lane 7). The blots were probed with Anti-FOXN3 Recombinant Rabbit Polyclonal Antibody (Product # 711585, 1-2 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 54 kDa band corresponding to FOXN3 protein was observed across the 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
- For immunofluorescence analysis, Hep G2 cells were fixed and permeabilized for detection of endogenous FOXN3 using Anti- FOXN3 Recombinant Rabbit Polyclonal Antibody (Product # 711585, 2 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 FOXN3 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 FOXN3. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Enrichment of endogenous FOXN3 protein at specific gene loci using Anti-FOXN3 Recombinant Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-FOXN3 Recombinant Rabbit Polyclonal Antibody (Product # 711585, 5 µg) on sheared chromatin from 2 million HEPG2 cells using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the different loci of the active MYC, PIM2 promoter region used as positive control target genes, and the region of the inactive SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
