NR2E3 Antibody, Biotin conjugated

Basic Research Questions

• What is NR2E3 and why is it important in retinal research?

  NR2E3, also known as Photoreceptor-specific nuclear receptor (PNR), is an orphan nuclear receptor that functions as a ligand-dependent transcription factor primarily expressed in retinal photoreceptor cells. It plays a crucial role as a transcriptional activator of rod development while simultaneously repressing cone development . NR2E3 binds to the promoter regions of several rod- and cone-specific genes, including rhodopsin, M- and S-opsin, and rod-specific phosphodiesterase beta subunit, where it enhances rhodopsin expression while repressing M- and S-cone opsin expression . The protein is expressed in late retinal progenitors and differentiating photoreceptors during development, and later localizes to the cell bodies of mature rods and cones . Its significance in retinal research stems from its involvement in multiple retinal degenerative diseases, including Enhanced S Cone Syndrome, making it an important target for both diagnostic and therapeutic applications.

• What are the key specifications of the NR2E3 Antibody, Biotin conjugated?

  The NR2E3 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbit against recombinant Photoreceptor-specific nuclear receptor protein (specifically amino acids 112-222) . It has been purified using Protein G, with purity exceeding 95% . The antibody is supplied in liquid form in a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . It has demonstrated reactivity with human samples and is primarily validated for ELISA applications . The UniProt ID for the target protein is Q9Y5X4, and the antibody recognizes NR2E3, which has a calculated molecular weight of 45 kDa and an observed molecular weight of 43-45 kDa .

  Specification	Details
  Antibody Type	Polyclonal
  Host	Rabbit
  Conjugate	Biotin
  Target	NR2E3 (amino acids 112-222)
  Reactivity	Human
  Applications	ELISA
  Purification Method	Protein G, >95%
  Storage Buffer	0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4
  Storage Conditions	-20°C or -80°C
  Target Molecular Weight	43-45 kDa

• What is the recommended storage protocol for NR2E3 Antibody, Biotin conjugated?

  The NR2E3 Antibody, Biotin conjugated should be stored at either -20°C or -80°C upon receipt . This low-temperature storage helps maintain the antibody's activity and specificity. It is crucial to avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of antibody function . The antibody is provided in a buffer containing 50% glycerol, which helps prevent freeze damage during storage. For laboratories that need to access the antibody frequently, it is advisable to prepare small aliquots before freezing to minimize freeze-thaw cycles. When handling the antibody, always use clean pipette tips and sterile techniques to prevent contamination, which could affect experimental outcomes and storage stability.

• What applications is the NR2E3 Antibody, Biotin conjugated optimized for?

  The NR2E3 Antibody, Biotin conjugated has been specifically tested and validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications . The biotin conjugation makes this antibody particularly suitable for sandwich ELISA methods where it can be used as a detection antibody in conjunction with streptavidin-HRP systems . In a typical sandwich ELISA workflow using this antibody, samples containing NR2E3 are first bound by an immobilized capture antibody pre-coated onto a microplate. After washing away unbound substances, the biotin-conjugated NR2E3 antibody is added to detect the bound NR2E3. Following another wash, streptavidin-conjugated HRP is added to bind to the biotin, and after a final wash, a substrate solution is added to produce color proportional to the amount of NR2E3 present in the sample .

• How does the biotin conjugation enhance the utility of NR2E3 antibodies in research?

  Biotin conjugation significantly enhances the utility of NR2E3 antibodies by leveraging the strong and specific interaction between biotin and streptavidin/avidin, which is one of the strongest non-covalent interactions known in biology. This conjugation offers several methodological advantages for researchers. First, it provides signal amplification capabilities, as multiple streptavidin molecules (conjugated to detection enzymes or fluorophores) can bind to each biotin molecule, thereby enhancing sensitivity in detection systems . Second, the biotin-streptavidin system offers flexibility in detection methods, allowing researchers to choose from various secondary detection reagents according to their specific experimental needs. Third, biotin conjugation enables more efficient washing steps in immunoassays like ELISA, reducing background signal and improving signal-to-noise ratios. The enhanced sensitivity and reduced background are particularly valuable when studying NR2E3, which may be expressed at relatively low levels in certain cell types or developmental stages.

Advanced Research Questions

• What methodological considerations are important when using NR2E3 Antibody, Biotin conjugated in sandwich ELISA systems?

  When implementing NR2E3 Antibody, Biotin conjugated in sandwich ELISA systems, several methodological considerations are critical for optimal results. First, antibody concentration must be carefully titrated—the recommended dilution range should be tested systematically in your specific system to determine optimal conditions for signal-to-noise ratio . Second, blocking buffers must be selected carefully to prevent non-specific binding while preserving NR2E3 epitope accessibility. Third, when designing the sandwich ELISA, ensure that the capture and detection antibodies recognize different, non-overlapping epitopes of NR2E3 to prevent competitive binding.

  For low abundance samples, consider implementing additional signal amplification strategies such as using poly-HRP streptavidin conjugates. Sample preparation is also crucial—use protease inhibitors during extraction to prevent NR2E3 degradation, and standardize protein concentration across samples. Finally, always include both positive controls (such as mouse or rat retina tissue extracts, which have confirmed NR2E3 expression) and negative controls (tissues known not to express NR2E3) to validate assay performance .

  Optimization Parameter	Recommendation
  Antibody Dilution	Test range 1:20-1:200 for optimal signal-to-noise ratio
  Positive Controls	Mouse retina tissue, Y79 cells, rat retina tissue
  Buffer System	PBS-based with minimal detergents
  Sample Preparation	Include protease inhibitors, standardize protein concentration
  Signal Development Time	Optimize based on standard curve to avoid saturation

• How can researchers validate the specificity of NR2E3 Antibody, Biotin conjugated in their experimental system?

  Validating the specificity of NR2E3 Antibody, Biotin conjugated requires a multi-faceted approach to ensure reliable experimental outcomes. Begin with positive control verification using tissues known to express NR2E3, such as mouse or rat retina tissues, Y79 cells, or HepG2 cells . These positive controls should demonstrate the expected 43-45 kDa band in Western blot applications or appropriate immunoreactivity in ELISA.

  For more rigorous validation, perform knockdown/knockout verification using cell lines with NR2E3 gene silencing through siRNA or CRISPR-Cas9 technology. The antibody signal should significantly decrease in these samples compared to wild-type controls. This approach has been documented in at least two publications referenced in the product information .

  Peptide competition assays can further confirm specificity—pre-incubation of the antibody with excess NR2E3 peptide (amino acids 112-222, as used in the immunogen) should block antibody binding and eliminate signal in subsequent assays. Additionally, cross-reactivity testing against related nuclear receptors from the same subfamily is recommended to ensure the antibody does not recognize similar epitopes in related proteins.

  Finally, compare results across multiple detection methods when possible. If NR2E3 expression patterns observed with the biotin-conjugated antibody align with those detected using other validated antibodies or mRNA expression data, this provides strong evidence for specificity.

• What protocol adaptations are necessary when using NR2E3 Antibody, Biotin conjugated to study retinal degenerative disorders?

  When employing NR2E3 Antibody, Biotin conjugated to study retinal degenerative disorders, several protocol adaptations are essential to accommodate the unique challenges of these disease models. First, temporal considerations are critical—NR2E3 expression and function change throughout disease progression, so establishing a detailed time course analysis is vital. In models like the Nr2e3rd7/rd7 mouse, sampling at multiple timepoints helps capture the progression from prolonged proliferation to apoptosis .

  Sample preparation requires special attention when working with degenerating retinas. The structural integrity of the tissue is often compromised, necessitating gentler fixation protocols and careful handling during processing. For ELISA applications, protein extraction methods may need optimization to account for altered cellular composition and potential changes in protein solubility.

  When analyzing results, consider the complex cellular changes occurring in degenerative conditions. In Nr2e3rd7/rd7 models, abnormal increases in cone photoreceptors arise from ectopic mitotic progenitor cells in the outer nuclear layer . This altered cellular composition affects the interpretation of quantitative measurements. Additionally, incorporate complementary techniques such as immunohistochemistry or in situ hybridization to correlate protein levels with tissue morphology and cell type-specific changes.

  For comparative studies, carefully select appropriate controls. Age-matched wild-type controls are essential, but also consider using models with different genetic causes of retinal degeneration to distinguish Nr2e3-specific effects from general degenerative processes. The temporal correlation between extended proliferation and pronounced apoptosis (~2:1 ratio of apoptotic to proliferating cells) observed at P30 in Nr2e3rd7/rd7 mice provides a critical reference point for such studies .

• How can NR2E3 Antibody, Biotin conjugated be integrated into studies of therapeutic interventions for retinitis pigmentosa?

  Integrating NR2E3 Antibody, Biotin conjugated into therapeutic intervention studies for retinitis pigmentosa (RP) requires strategic experimental design that leverages NR2E3's role as both a disease-causing gene and a potential modifier gene therapeutic. Recent research demonstrates that NR2E3 can attenuate retinal degeneration in RhoP23H mouse models, suggesting its potential as a therapeutic agent . When designing such studies, implement a longitudinal approach with multiple timepoints to track therapy effects, as demonstrated in the six-month follow-up evaluations of different NR2E3 doses in heterozygous Rho mouse models .

  For quantitative assessment of therapeutic efficacy, use the antibody in ELISA applications to measure NR2E3 protein levels across treatment groups, correlating these with functional outcomes measured by electroretinogram (ERG) and histological preservation. The antibody can help determine if exogenous NR2E3 expression reaches therapeutic levels and persists throughout the treatment period.

  Critical controls must include age-matched wild-type animals, untreated disease models, and possibly animals treated with control vectors lacking therapeutic genes. When analyzing therapeutic outcomes, examine the correlation between NR2E3 levels and the regulation of downstream pathways, including ER stress response, neuroprotection, apoptosis, immune response, and cell survival mechanisms .

  Assessment Parameter	Methodology	Timeline
  NR2E3 Protein Levels	ELISA using biotin-conjugated antibody	Baseline, 1, 3, 6 months
  Retinal Function	Electroretinogram (ERG)	Baseline, 1, 3, 6 months
  Photoreceptor Preservation	Histology and immunohistochemistry	End of study (6 months)
  Downstream Pathway Regulation	Multiplex protein analysis	Mid and end points

• What strategies can overcome potential limitations when using NR2E3 Antibody, Biotin conjugated in complex retinal samples?

  When working with complex retinal samples, researchers face several challenges that require specific strategies to optimize the performance of NR2E3 Antibody, Biotin conjugated. First, address sample heterogeneity by implementing laser capture microdissection to isolate specific retinal layers or cell populations before analysis, ensuring more precise quantification of NR2E3 in relevant cell types. This is particularly important given that NR2E3 is expressed in both late retinal progenitors and mature photoreceptors .

  To overcome potential epitope masking from tissue fixation or processing, optimize antigen retrieval methods. For retinal samples, consider using TE buffer (pH 9.0) as suggested for immunohistochemistry applications with related NR2E3 antibodies, or alternatively, citrate buffer (pH 6.0) . Test both methods to determine which provides optimal epitope accessibility without compromising tissue integrity.

  For samples with low NR2E3 expression, implement signal amplification systems. While the biotin-streptavidin system already offers amplification, further sensitivity can be achieved using tyramide signal amplification (TSA) compatible with biotin-conjugated antibodies. This can be particularly valuable when studying early disease stages or examining cell populations with naturally low NR2E3 expression.

  To address potential non-specific binding in retinal tissue, which contains high lipid content, modify blocking buffers by including additional blocking agents such as normal serum from the same species as secondary reagents, milk proteins, or commercial protein-free blocking buffers. Additionally, increase the stringency of wash steps by adjusting salt concentration or adding low concentrations of non-ionic detergents.

  Finally, implement multiplexing strategies to simultaneously detect NR2E3 along with cell-type specific markers (rhodopsin for rods, opsins for cones) to provide context for NR2E3 expression patterns and overcome interpretational limitations from complex tissue architecture.

• How does NR2E3 function as a modifier gene in retinal degeneration, and how can the biotin-conjugated antibody help elucidate these mechanisms?

  NR2E3 functions as a crucial modifier gene in retinal degeneration through multiple mechanisms that can be investigated using the biotin-conjugated NR2E3 antibody. As a transcription factor, NR2E3 regulates gene networks involved in ER stress, neuroprotection, photoreceptor function, apoptosis, immune response, and cell survival pathways . The variable phenotypes observed in NR2E3-associated retinal degenerations suggest that NR2E3 interacts with other genetic factors to influence disease manifestation and progression .

  The biotin-conjugated antibody can be employed in chromatin immunoprecipitation followed by sequencing (ChIP-seq) studies to map NR2E3 binding sites across the genome in different retinal degeneration models. This approach would identify direct transcriptional targets that mediate its modifier effects. In ELISA-based protein-protein interaction studies, the antibody can help identify cofactors that associate with NR2E3 in healthy versus degenerating retinas.

  Longitudinal studies tracking NR2E3 protein levels during disease progression are particularly informative. In preclinical models, three different doses of NR2E3 demonstrated efficacy in reducing retinal degeneration and improving retinal morphology over six months . The biotin-conjugated antibody can quantitatively assess NR2E3 levels in these dose-response studies, correlating protein expression with functional outcomes measured by ERG and histological preservation.

  Importantly, recent research shows that NR2E3 can attenuate retinal degeneration in RhoP23H mouse models even though the primary mutation is not in NR2E3 . This suggests NR2E3's potential as a broad-spectrum therapeutic agent, particularly valuable in RP cases where the primary mutation cannot be identified. The biotin-conjugated antibody provides a sensitive tool for monitoring therapeutic NR2E3 levels in such intervention studies.

• What role does NR2E3 play in photoreceptor cell fate determination, and how can this be studied using the biotin-conjugated antibody?

  NR2E3 plays a pivotal role in photoreceptor cell fate determination as both an activator of rod development and a repressor of cone development . Studies using Nr2e3rd7/rd7 mice reveal that NR2E3 functions in late retinal progenitors to suppress cone cell generation . The abnormal increase in cone photoreceptors observed in these mice arises from ectopic mitotic progenitor cells present in the outer nuclear layer of the mature retina .

  The biotin-conjugated NR2E3 antibody can be instrumental in studying this process through several methodological approaches. In developmental time-course studies, the antibody can be used in ELISA assays to quantify NR2E3 protein levels at different stages of retinal development, correlating expression patterns with photoreceptor specification events. This is particularly relevant since NR2E3 acts simultaneously in different cell types: late mitotic progenitors, newly differentiating post-mitotic cells, and mature rods and cones .

  For mechanistic investigations, the antibody can be employed in co-immunoprecipitation experiments to identify protein interaction partners that mediate NR2E3's dual function in activating rod genes while repressing cone genes. When coupled with subsequent proteomic analysis, this approach can reveal cell-type specific protein complexes that contribute to photoreceptor fate decisions.

  Researchers can also use the antibody to examine the relationship between NR2E3 expression and cell proliferation. In Nr2e3rd7/rd7 mice, a prolonged phase of proliferation is observed, followed by abnormal retinal lamination . By combining the NR2E3 antibody with proliferation markers in flow cytometry or immunohistochemistry, researchers can track how NR2E3 levels correlate with cell cycle exit and differentiation commitment.

  Development Stage	NR2E3 Expression	Function	Cellular Context
  Late retinal progenitors	Expressed	Suppresses cone generation program	Mitotic cells
  Differentiating photoreceptors	Expressed	Promotes rod development	Post-mitotic cells
  Mature photoreceptors	Localized to cell bodies	Maintains photoreceptor identity	Terminally differentiated cells