NR2E3 Antibody, HRP conjugated

What is NR2E3 and why is it an important target for research?

NR2E3, also known as Photoreceptor-Specific Nuclear Receptor (PNR), is a ligand-dependent transcription factor that belongs to the nuclear receptor family. It plays a crucial role in retinal development and photoreceptor differentiation, particularly in rod photoreceptors. NR2E3 functions as a transcriptional regulator that suppresses cone-specific gene expression while activating rod-specific genes . The importance of NR2E3 in research stems from its role in retinal diseases - genetic mutations in NR2E3 are associated with enhanced S-cone syndrome, Goldman-Favre syndrome, and retinitis pigmentosa. Research targeting NR2E3 is valuable for understanding photoreceptor development, differentiation, and potential therapeutic approaches for retinal degeneration disorders .

What applications is NR2E3 Antibody, HRP conjugated suitable for?

NR2E3 Antibody, HRP conjugated is particularly well-suited for applications requiring direct detection without secondary antibodies. Based on data from similar NR2E3 antibodies, the HRP-conjugated version is appropriate for:

For Western blotting applications, the antibody can detect bands corresponding to the observed molecular weight of 43-45 kDa, consistent with the calculated molecular weight of 45 kDa for NR2E3 .

What species reactivity has been validated for NR2E3 Antibody?

The reactivity profile for NR2E3 antibodies has been extensively tested across multiple species:

Positive Western blot detection has been specifically confirmed in mouse retina tissue, Y79 cells, HepG2 cells, and rat retina tissue . For immunohistochemistry applications, positive staining has been observed in human prostate cancer tissue .

What are the recommended dilutions for different applications of NR2E3 Antibody, HRP conjugated?

Optimal dilution ranges for NR2E3 antibodies vary by application type. For the HRP-conjugated version, researchers should consider these starting dilutions, adjusting based on signal intensity and background:

It is strongly recommended that each research group titrate the antibody in their specific experimental system to determine optimal conditions . The wide dilution range for Western blotting (1:2000-1:12000) indicates that the antibody has good sensitivity and can be highly diluted in many applications while maintaining specific signal.

What buffer systems and antigen retrieval methods work best with NR2E3 Antibody?

For optimal results with NR2E3 antibody in immunohistochemistry applications:

• Primary antigen retrieval recommendation: TE buffer pH 9.0

• Alternative approach: Citrate buffer pH 6.0

For Western blotting, the antibody performs well in standard TBS-T or PBS-T buffer systems with 5% non-fat dry milk or BSA as blocking agents.

Storage recommendations for preserving antibody activity:

• Store at -20°C in PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Stable for one year after shipment when stored properly

• Aliquoting is unnecessary for -20°C storage

• Smaller (20μl) sizes may contain 0.1% BSA for stabilization

How can I validate the specificity of NR2E3 Antibody, HRP conjugated in my experimental system?

Multiple approaches can be employed to validate antibody specificity:

• Positive controls: Use tissues/cells known to express NR2E3, such as:

  • Mouse or rat retina tissue

  • Y79 retinoblastoma cells

  • Photoreceptor progenitor cells

  • HepG2 cells

• Knockdown/knockout validation: Several publications have documented NR2E3 antibody specificity using KD/KO systems, confirming signal reduction or elimination in these models .

• Molecular weight verification: Confirm detection at the expected molecular weight of 43-45 kDa .

• Peptide competition: Pre-incubation with immunizing peptide should abolish specific signals.

• Cross-reactivity assessment: Test in samples from multiple species to confirm the expected reactivity pattern.

How can NR2E3 Antibody, HRP conjugated be utilized in studies of rod photoreceptor development?

NR2E3 plays a critical role in rod photoreceptor development and differentiation. Research has shown that genetic deletion of Nr2e3 can reprogram rods into cells resembling cone photoreceptors . When designing experiments to study this process:

• Developmental time-course studies: Use the antibody to track NR2E3 expression during different stages of retinal development

• Co-localization studies: Combine with markers for rod (rhodopsin) and cone photoreceptors to examine cell fate determination

• Transcription factor interactions: NR2E3 functions in a network with other transcription factors like NRL and CRX. Co-immunoprecipitation experiments can investigate protein-protein interactions:

  • Transfect cells with hNRL-pCMVSport6, hCRX-pCMVSport6, and hNR2E3-pcDNA3.1/HisC

  • Lyse cells with appropriate Co-IP buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5% glycerol, with protease inhibitors)

  • Perform standard Co-IP protocols followed by Western blot detection with the HRP-conjugated NR2E3 antibody

• Chromatin immunoprecipitation: Examine NR2E3 binding to regulatory regions of rod- and cone-specific genes

What methodological approaches can resolve inconsistent results when using NR2E3 Antibody, HRP conjugated?

When troubleshooting inconsistent results with NR2E3 Antibody, HRP conjugated, consider these methodological approaches:

• Epitope accessibility: The antibody is generated against NR2E3 fusion protein Ag5503 . Post-translational modifications or protein-protein interactions may mask the epitope. Consider:

  • Alternative sample preparation methods

  • Denaturing versus non-denaturing conditions

  • Multiple antigen retrieval approaches for IHC

• Signal enhancement strategies:

  • For weak signals, employ tyramide signal amplification (TSA) to amplify HRP signal

  • Optimize incubation time and temperature

  • Test enhanced chemiluminescent substrates with different sensitivities

• Background reduction:

  • Implement more stringent blocking (5% BSA or commercial blocking reagents)

  • Include 0.1% Tween-20 in antibody diluent

  • For tissues with high endogenous peroxidase activity, include peroxidase quenching step (3% H₂O₂) prior to antibody incubation

• Sample-specific optimization:

  • For retinal samples: Specialized fixation protocols may better preserve epitopes in photoreceptor cells

  • For cultured cells: Compare results between different fixation methods (4% PFA, methanol, acetone)

How can small molecule modulators of NR2E3 function be studied using this antibody?

High-throughput screening approaches have identified potential small molecule modulators of NR2E3 function . When investigating these compounds:

• Expression level analysis: Western blotting with NR2E3 Antibody, HRP conjugated can determine if small molecules alter NR2E3 protein levels

• Protein interaction studies:

  • Small molecules may disrupt NR2E3 interactions with co-repressors like Ncor

  • Co-immunoprecipitation followed by Western blotting can detect changes in protein complex formation

  • Experimental approach: Transfect cells with NR2E3 constructs, treat with compounds, perform Co-IP and blotting

• Target gene expression:

  • NR2E3 regulates rhodopsin expression in rod photoreceptors

  • After compound treatment, measure changes in rhodopsin levels by Western blot or immunostaining

  • Cell-based assays can include dissociated retinal cells from P5 wild-type mice treated with compounds at 1 μM for 3 days

• Chromatin occupancy:

  • ChIP assays using the HRP-conjugated antibody can determine if small molecules alter NR2E3 binding to target gene promoters

  • This provides mechanistic insights into compound activity

What are the critical factors for optimizing signal-to-noise ratio with NR2E3 Antibody, HRP conjugated?

Optimizing signal-to-noise ratio is essential for generating reliable data with HRP-conjugated antibodies:

• Antibody concentration optimization:

  • The wide dilution range (1:2000-1:12000 for WB) suggests starting with higher dilutions and adjusting as needed

  • Titration experiments are essential for each new tissue/cell type

• Blocking optimization:

  • For Western blotting: Compare 5% non-fat milk versus 5% BSA in TBS-T

  • For IHC/ICC: Test commercial blocking reagents versus serum-based blockers from species unrelated to host (rabbit IgG)

• Incubation conditions:

  • Compare room temperature (1-2 hours) versus 4°C overnight incubation

  • For IHC with enhanced signals, shorter incubation times may reduce background

• Detection substrate selection:

  • For Western blotting: Standard ECL for strong signals, femto-level substrates for low abundance targets

  • For IHC: DAB concentration and development time critically impact signal-to-noise ratio

• Wash stringency:

  • Increase number of washes (5-6 washes instead of standard 3)

  • Extended wash duration (10 minutes per wash) may improve results with difficult samples

How does sample preparation affect detection efficiency for NR2E3 in different tissue types?

Sample preparation significantly impacts NR2E3 detection across different tissues:

• Retinal tissue preparation:

  • Fresh frozen sections generally preserve epitopes better than paraffin-embedded samples

  • For fixed tissues, short fixation times (4-6 hours) in 4% PFA yield optimal results

  • Cryoprotection with sucrose gradients before freezing improves tissue morphology

• Cultured cell preparation:

  • For adherent cells: Direct lysis on plate yields better results than scraping/pelleting

  • For Y79 suspension cells: Gentle centrifugation (300g) prevents cell damage and protein degradation

• Sample buffer considerations for Western blotting:

  • RIPA buffer extraction yields strong NR2E3 signals

  • Include protease inhibitors to prevent degradation

  • Sample heating at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol effectively denatures NR2E3

• Extraction method comparison for nuclear proteins:

  • As a transcription factor, NR2E3 is primarily nuclear

  • Nuclear extraction protocols yield enriched samples with reduced cytoplasmic contamination

  • Compare commercial nuclear extraction kits versus differential centrifugation methods

What quantitative analytical approaches are appropriate when using NR2E3 Antibody, HRP conjugated?

For quantitative analysis with HRP-conjugated NR2E3 antibody:

• Western blot densitometry:

  • Include appropriate loading controls (β-actin, GAPDH for total protein; Lamin B, Histone H3 for nuclear fractions)

  • Use software like ImageJ for quantification

  • Generate standard curves with recombinant protein for absolute quantification

  • Stay within linear range of detection for accurate quantification

• Immunohistochemistry quantification:

  • For DAB staining: Use image analysis software with color deconvolution algorithms

  • Account for regional variations in expression (especially in retinal tissue)

  • Standardize image acquisition parameters across experimental groups

• ELISA development:

  • Sandwich ELISA protocols can be developed using capture and HRP-conjugated detection antibodies

  • Generate standard curves with recombinant NR2E3 protein

  • Optimize plate coating, blocking, and washing steps for consistent results

• Statistical considerations:

  • Perform replicate experiments (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Account for batch effects in multi-day experiments

How can NR2E3 Antibody, HRP conjugated contribute to retinal disease research and therapeutic development?

NR2E3 mutations are implicated in several retinal diseases, making this antibody valuable for disease research:

• Disease mechanism studies:

  • Compare NR2E3 expression and localization in normal versus diseased retinal tissue

  • Examine downstream effects on rod/cone-specific gene networks

  • Investigate interactions with other disease-associated proteins

• Therapeutic screening platforms:

  • High-throughput cell-based assays using the antibody can screen for compounds that modulate NR2E3 activity

  • Previous screens have utilized TR-FRET biochemical assays and cell-based luminescence assays to identify active compounds

  • Promising compounds can be tested in dissociated retinal cell cultures and animal models

• Gene therapy monitoring:

  • For gene replacement strategies, the antibody can verify expression of the therapeutic NR2E3 construct

  • Western blotting and immunohistochemistry provide complementary information on expression levels and localization

• Small molecule therapeutic development:

  • The identification of compounds that modulate Nr2e3-Ncor interactions suggests potential therapeutic approaches

  • The HRP-conjugated antibody can validate target engagement in cell and tissue models

What multiplex experimental designs can incorporate NR2E3 Antibody, HRP conjugated?

Advanced multiplex approaches can maximize data from limited samples:

• Sequential immunodetection on Western blots:

  • Detect NR2E3 first using the HRP-conjugated antibody

  • Strip membrane and reprobe for related transcription factors (NRL, CRX)

  • Document complete stripping by incubating with detection reagent prior to reprobing

• Multiplex immunohistochemistry:

  • Tyramide signal amplification (TSA) approaches allow multiple antigens on the same section

  • After HRP detection, perform heat-mediated antibody stripping

  • Proceed with subsequent primary antibodies to reveal co-expression patterns

• Combined techniques:

  • RNA in situ hybridization followed by protein immunodetection

  • This approach correlates mRNA expression with protein levels within the same cells/tissues

  • Particularly valuable for studying transcriptional regulation by NR2E3

• Mass cytometry approaches:

  • Metal-conjugated antibodies enable highly multiplexed single-cell analysis

  • Custom conjugation of anti-NR2E3 to rare earth metals allows integration into CyTOF panels

The implementation of these advanced research strategies will continue to expand our understanding of NR2E3 biology and its role in retinal development and disease processes.