NR2E3 Antibody, FITC conjugated

What is NR2E3 and why is it significant in retinal research?

NR2E3 (Nuclear Receptor Subfamily 2 Group E Member 3), also known as Photoreceptor-Specific Nuclear Receptor (PNR), belongs to a large family of nuclear hormone receptor transcription factors. These proteins feature discrete domains that function in DNA and ligand binding. NR2E3 plays a pivotal role in regulating signaling pathways essential for photoreceptor cell function and embryonic development. It is an eye-specific nuclear protein localized to the outer nuclear layer of the adult retina, where the nuclei of cone and rod photoreceptors reside. The protein serves as a transcriptional activator of rod development while simultaneously repressing cone development. Defects in the NR2E3 gene, which maps to chromosome 15q22.32, are associated with enhanced S cone syndrome and other retinopathies . Understanding NR2E3 function is critical for elucidating mechanisms of photoreceptor differentiation and potential therapeutic interventions for retinal degenerative diseases.

What are the recommended dilutions and applications for FITC-conjugated NR2E3 antibodies?

According to manufacturer specifications, FITC-conjugated NR2E3 antibodies are suitable for various applications with specific recommended dilutions. For immunofluorescence on paraffin-embedded tissues (IF/IHC-P), the recommended dilution range is 1:50-200 . For ELISA and Dot Blot applications, the antibody has demonstrated reliable performance, though specific dilution recommendations may vary based on the particular assay setup . When using this antibody for novel applications or in different experimental systems, titration experiments are advisable to determine optimal working concentrations. Applications should be validated with appropriate positive and negative controls, including tissues known to express NR2E3 (such as retinal tissue) and non-expressing tissues or cells. For immunofluorescence applications, common positive controls include mouse or human retina tissues, while Y79 retinoblastoma cells and HepG2 cells have also shown detectable expression of NR2E3 .

How can I validate the specificity of NR2E3 antibody in experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For NR2E3 antibodies, several approaches can be implemented. First, conduct western blot analysis using retinal tissue lysates from human, mouse, or rat samples, which have been confirmed as reactive species for many commercial NR2E3 antibodies . The expected molecular weight for human NR2E3 is approximately 41 kDa. Second, perform immunohistochemistry on retinal sections, where specific staining should be observed in the outer nuclear layer containing photoreceptor nuclei. Compare staining patterns with published literature on NR2E3 localization. Third, include appropriate controls in your experiments: (1) positive controls using tissues known to express NR2E3, such as retina samples; (2) negative controls by omitting primary antibody; and (3) if possible, tissues from NR2E3 knockout models (such as rd7/rd7 mice) or cells with CRISPR-mediated NR2E3 knockout. For RNA interference studies, correlate protein reduction via immunostaining with successful NR2E3 mRNA knockdown. Finally, peptide competition assays can be performed by pre-incubating the antibody with the immunizing peptide before application to samples, which should abolish specific staining .

What experimental considerations are important when using NR2E3 antibodies in chromatin immunoprecipitation (ChIP) assays?

When performing ChIP assays with NR2E3 antibodies, several critical factors should be considered to ensure successful experiments. Based on previous studies with NR2E3, cross-linking conditions typically involve 1-2% formaldehyde for 10-15 minutes at room temperature. For sonication, conditions should be optimized to generate DNA fragments of approximately 200-500 bp. In previously published protocols, chromatin was immunoprecipitated with NR2E3 antibody and subsequently washed with low-salt, high-salt, and LiCl wash buffers before reverse cross-linking with NaCl (200 mM final concentration) and incubation with RNase A at 65°C for five hours . Appropriate controls include: (1) input samples (non-immunoprecipitated chromatin); (2) IgG control (using matched isotype IgG from the same species as the NR2E3 antibody); and (3) positive control regions known to be bound by NR2E3, such as promoters of rhodopsin, M- and S-opsin genes . PCR primers should be designed to amplify approximately 200 bp products from regions containing putative NR2E3 response elements. Reactions typically employ 35 cycles with an annealing temperature of 58°C, followed by electrophoresis in 2% agarose gel and visualization with ethidium bromide staining .

How does the performance of NR2E3 antibodies vary across different species' retinal tissues?

Commercial NR2E3 antibodies demonstrate variable cross-reactivity across species, which is an important consideration for comparative studies. According to product specifications, the FITC-conjugated rabbit polyclonal NR2E3 antibody shows reactivity with human, mouse, and rat samples . Another rabbit polyclonal antibody preparation is specified as reactive only with human samples . Western blot detection has been confirmed in mouse retina tissue, rat retina tissue, Y79 human retinoblastoma cells, and HepG2 human hepatocellular carcinoma cells . The differential reactivity is likely due to variations in epitope conservation across species. When working with non-validated species, preliminary validation experiments are essential. For cross-species studies, researchers should select antibodies with confirmed reactivity across the species of interest or validate the antibody in each species individually. Sequence homology analysis of the immunogen region (for example, amino acids 112-222 of human NR2E3 used in some commercial antibodies ) against the target species' NR2E3 sequence can provide a theoretical prediction of cross-reactivity potential. For immunohistochemistry applications in comparative studies, consistent fixation methods and antigen retrieval protocols should be employed across all samples to minimize technique-induced variability .

What are the optimal protocols for co-localization studies using FITC-conjugated NR2E3 antibodies?

For co-localization studies combining FITC-conjugated NR2E3 antibodies with other markers, careful experimental design is essential to minimize spectral overlap while maximizing signal detection. Based on published protocols, retinal sections should be fixed with either 4% paraformaldehyde or methanol/acetic acid (3:1), maintaining dorsal/ventral orientation for consistent anatomical reference . When selecting additional markers, avoid fluorophores with significant spectral overlap with FITC (excitation ~495 nm, emission ~519 nm). Compatible combinations include: (1) FITC-conjugated NR2E3 with rhodopsin antibody detected by Alexa Fluor 594/Texas Red secondary antibody for rod photoreceptor co-localization; (2) FITC-conjugated NR2E3 with peanut agglutinin (typically visualized with far-red fluorophores) for cone photoreceptor association studies . After blocking with 2% normal serum, incubate sections with FITC-conjugated NR2E3 antibody (1:50-200 dilution) and the unconjugated primary antibody for the second marker overnight at 4°C. The following day, apply only the secondary antibody for the unconjugated primary. For optimal results, use sequential imaging rather than simultaneous acquisition of different channels to minimize potential bleed-through artifacts. Include single-labeled controls to verify absence of spectral overlap and false positive co-localization. Confocal microscopy with appropriate bandwidth settings is recommended for definitive co-localization analysis .

How can NR2E3 antibodies be utilized to investigate the molecular mechanisms of enhanced S-cone syndrome (ESCS)?

Enhanced S-cone syndrome (ESCS) and related retinopathies result from mutations in the NR2E3 gene, making NR2E3 antibodies valuable tools for investigating disease mechanisms. A comprehensive approach utilizing these antibodies might include several strategies. First, immunohistochemical comparison between normal and ESCS patient retinal samples using FITC-conjugated NR2E3 antibodies can reveal alterations in protein localization and expression levels. In normal retina, NR2E3 localizes to the outer nuclear layer, while altered patterns may be observed in ESCS tissues . Second, analysis of NR2E3-related transcriptional networks can be performed using chromatin immunoprecipitation followed by sequencing (ChIP-seq) or targeted ChIP-PCR. Previous studies identified several NR2E3 target genes including transcription factors (Ror1, Rorg) and nuclear hormone receptors (Nr1d1, Nr2c1) during development, and rod-specific genes like Gnb1 in mature retina . Researchers can assess if NR2E3 mutations affect binding to these targets. Third, co-immunoprecipitation studies using NR2E3 antibodies can investigate protein-protein interactions potentially disrupted in ESCS. Fourth, the rd7/rd7 mouse model, which exhibits a phenotype similar to human ESCS with hybrid photoreceptors expressing both rod and cone genes, can be examined using immunofluorescence to detect alterations in photoreceptor markers alongside NR2E3 . Comparative RNA sequencing of wild-type versus rd7/rd7 retinas, coupled with NR2E3 ChIP data, can identify direct versus indirect transcriptional effects. Finally, rescue experiments in rd7/rd7 mice or patient-derived induced pluripotent stem cells with wild-type NR2E3 can be monitored using the antibody to confirm restoration of proper protein expression and localization .

What approaches can be used to optimize the use of NR2E3 antibodies in time-resolved fluorescence energy transfer (TR-FRET) assays for drug discovery?

TR-FRET assays represent a sophisticated application of NR2E3 antibodies for high-throughput screening (HTS) of potential therapeutic modulators. Based on published protocols, several optimization strategies can enhance assay performance. First, ensure high-quality protein preparation by using a purification protocol for functionally competent soluble NR2E3 protein expressed in insect Sf9 cells, as this has proven successful in previous studies . For the TR-FRET assay design, consider the established model measuring agonist-sensitive interaction between apo-NR2E3 and transcriptional corepressor RetCOR. This assay utilizes GST-tagged NR2E3 and MBP-tagged RetCOR fragment, detecting their interaction through appropriate fluorophore-labeled antibodies . When adapting a FITC-conjugated NR2E3 antibody for this purpose, modifications may be necessary depending on the emission/excitation parameters of other assay components. For assay optimization, the following parameters should be considered: (1) protein concentration ratios (NR2E3:RetCOR optimal ratios determined through titration); (2) incubation time and temperature (typically room temperature for 1-2 hours); (3) buffer composition (including potential additives like DTT, detergents, or stabilizers); and (4) DMSO tolerance (important for compound solubilization). For positive control development, since NR2E3 is an orphan nuclear receptor without known ligands, consider using 10 μM biotin (>1,000-fold molar excess over biotinylated protein) to mimic the effect of an agonist by preventing protein-protein interaction . Miniaturization to 1,536-well format has been successful for this assay type, requiring only three pipetting steps. For quality control, aim for Z'-scores within the 0.6-0.8 range to ensure reliable HTS performance. Finally, include appropriate counterscreen assays, such as measuring the effect of test compounds on PPARγ interaction with corepressor NCOR, to assess compound specificity .