Nrl Antibody

What is NRL and why is it important in retinal research?

NRL is a 30 kDa nuclear transcription factor belonging to the Maf subfamily within the bZIP (basic motif-leucine zipper) transcription factor family. It contains an N-terminal transcriptional activation domain and a C-terminal DNA-binding bZIP domain that facilitates homo- or heterodimerization . NRL is critical in vision research because it:

• Functions as a master regulator of rod photoreceptor cell fate in the retina

• Regulates the expression of rod-specific genes

• Is expressed specifically in rod photoreceptors

• Acts as a transcriptional activator for genes like RHO and PDE6B

• Functions as a transcriptional coactivator, stimulating transcription mediated by CRX and NR2E3

NRL's importance is further underscored by its role in retinal diseases, as mutations are associated with retinitis pigmentosa and other retinal degenerative disorders .

What species reactivity can I expect from commercially available NRL antibodies?

Current commercially available NRL antibodies show diverse reactivity profiles:

When selecting an antibody, consider your experimental species and application requirements. Most commercial antibodies demonstrate high sequence conservation across species, with human NRL sharing 92%, 94%, 90%, and 90% sequence identity with bovine, canine, rat, and mouse NRL, respectively .

What is the molecular structure and post-translational modification profile of NRL?

NRL protein exhibits several important structural and post-translational characteristics:

• Calculated molecular weight: ~26 kDa (237 amino acids)

• Observed molecular weight: ~30 kDa on Western blots

• Post-translational modifications:

  • Phosphorylation at multiple sites, likely regulating activity

  • Disumoylation at Lys-20, which modulates transcriptional activity on RHO and NR2E3 promoters

  • SUMOylation site (K20) is conserved across species

On immunoblots, NRL typically appears as a characteristic doublet band due to these post-translational modifications, particularly SUMOylation .

What are the optimal dilutions and protocols for using NRL antibodies in different applications?

Based on manufacturer recommendations and published studies:

For immunohistochemistry applications, detecting NRL in retinoblastoma tissue has been validated . When using anti-NRL antibodies for ChIP experiments, shear chromatin to 200-500 bp fragments for optimal results .

How can I effectively use NRL antibodies in ChIP assays to study transcriptional regulation?

ChIP assays using NRL antibodies have been effectively employed to study NRL's direct regulation of target genes. A recommended protocol based on published research includes:

• Cross-link retinal tissue with 1% formaldehyde (15 min at room temperature)

• Stop reaction with glycine (0.125 M final concentration)

• Mechanically dissociate tissue and lyse with 0.5% Nonidet P-40

• Lyse nuclear pellet with 1% SDS and 0.5% EmpigenBB

• Sonicate to achieve chromatin fragments of 200-500 bp

• Immunoprecipitate with 2.5 μg anti-NRL antibody overnight at 4°C

• Use protein G Dynabeads for antibody capture (2 h at 4°C)

• Include rabbit IgG controls

• Design primers to amplify regions containing potential NRL binding sites

This approach has successfully identified direct binding of NRL to promoters of genes involved in photoreceptor development and function, including binding to NRE (NRL response elements) in Thrb and S-opsin promoters .

How can I optimize immunofluorescence detection of NRL in retinal tissues?

For effective immunofluorescence detection of NRL:

• Fixation: 4% PFA fixation of eyeballs followed by careful retinal dissection

• Processing: Subject retinas to sucrose gradient series (5%, 15%, 30%)

• Sectioning: Embed in OCT compound and create cryosections

• Antibody incubation: Optimize primary antibody dilution (typically 1:500 for commercial antibodies)

• Controls: Include appropriate controls:

  • Positive control: Wild-type retina sections

  • Negative control: Nrl knockout retina when available

  • Secondary antibody-only control

For co-localization studies, NRL has been successfully co-stained with rod-specific markers like rhodopsin (RHO) and rod-specific transcription factors like NR2E3, while confirming absence of co-localization with cone-specific markers like ARR3 .

How can NRL antibodies be used to study retinal development and disease models?

NRL antibodies have proven valuable for studying developmental processes and disease mechanisms:

• Developmental studies:

  • Track rod photoreceptor differentiation in developing retinas

  • Monitor temporal expression patterns during neurogenesis

  • In reporter lines like NRL+/eGFP hESC, antibodies confirm reporter specificity

• Disease models:

  • Evaluate rod photoreceptor preservation in retinal degeneration models

  • Study photoreceptor rescue following gene therapy interventions

  • Analyze Nrl expression following CRISPR/Cas9-mediated gene editing in models like Pde6a mice

For example, in research examining retinal degeneration in Pde6a mice, NRL antibodies (Proteintech #17388-1-AP) were used to evaluate NRL expression following gene inactivation therapy, confirming successful manipulation of photoreceptor cell fate as a therapeutic strategy .

How can I use NRL antibodies to investigate protein-protein interactions in transcriptional complexes?

NRL functions within complex transcriptional networks involving interactions with other key transcription factors. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-NRL antibodies to pull down NRL and its binding partners

  • Detect interaction with factors like CRX and NR2E3 by Western blot

  • Include appropriate controls (IgG, lysate input)

• ChIP-reChIP:

  • First immunoprecipitate with anti-NRL antibody

  • Then perform a second immunoprecipitation with antibodies against suspected interacting factors

  • This identifies genomic regions where both factors co-bind

• Proximity Ligation Assay (PLA):

  • Use anti-NRL antibody alongside antibodies against potential interacting partners

  • Visualize protein-protein interactions in situ with subcellular resolution

These approaches have helped establish that NRL interacts with CRX and NR2E3 to regulate gene expression in rod photoreceptors .

What role does NRL play in retinal organoid development, and how can antibodies track this process?

NRL antibodies have been instrumental in characterizing photoreceptor development in retinal organoids:

• Temporal expression patterns:

  • In NRL+/eGFP hESC-derived organoids, NRL expression begins around day 70 of differentiation

  • By day 130, approximately 50% of cells express NRL

  • The most dramatic increase occurs between days 70-95

• Structural development:

  • By day 145, NRL antibody staining reveals a distinct ONL-like layer

  • NRL+ cells co-express rod-specific markers like NR2E3 and rhodopsin

  • This allows tracking of outer segment development and synapse formation

• Quantification methods:

  • Dissociated organoids can be immunostained for NRL and quantified using high content image analysis

  • This approach reveals that approximately 47-50% of cells adopt rod identity by day 130

Why might I observe a doublet band when detecting NRL by Western blot?

The characteristic doublet pattern observed in NRL Western blots has been documented across species and is attributed to post-translational modifications:

• SUMOylation: The primary cause of the doublet is SUMOylation at lysine 20 (K20)

  • This site is conserved across species, including zebrafish, mouse, and human

  • SUMOylation modulates NRL's transcriptional activity on target promoters

• Phosphorylation: NRL is phosphorylated at multiple sites, which can also affect migration

  • Phosphorylation likely regulates NRL activity

  • Treatment with phosphatases can help confirm this modification

To confirm that the doublet represents genuine NRL protein:

• Include positive controls (wild-type retinal lysate)

• Include negative controls (Nrl knockout retinal lysate when available)

• Consider phosphatase or deSUMOylation treatments to observe band shifts

What antigen retrieval methods work best for immunohistochemical detection of NRL?

Effective antigen retrieval is critical for NRL detection in fixed tissues:

• Recommended methods:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative approach)

• Tissue preparation:

  • For frozen sections: Fix in 4% PFA, process through sucrose gradient

  • For paraffin sections: Additional optimization of retrieval time may be necessary

• Blocking and background reduction:

  • Use 0.5% BSA in blocking solutions

  • Include 0.1-0.3% Triton X-100 for permeabilization

  • Consider using species-specific serum that matches secondary antibody

For human retinoblastoma tissue specifically, TE buffer pH 9.0 has been validated for successful NRL detection .

How can I validate the specificity of my NRL antibody results?

Rigorous validation is essential for confident interpretation of NRL antibody results:

• Genetic controls:

  • Nrl knockout tissue/cells serve as ideal negative controls

  • Tissue with known high NRL expression (retina) as positive control

  • Comparative analysis across species to verify evolutionary conservation

• Technical controls:

  • Secondary antibody-only controls to assess background

  • Isotype controls (rabbit IgG) for non-specific binding

  • Blocking peptide competition to confirm epitope specificity

• Cross-validation approaches:

  • Compare results from multiple anti-NRL antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Verify results using reporter lines (e.g., NRL+/eGFP)

Well-documented cross-validation approaches include comparing NRL immunolabeling with eGFP expression in NRL+/eGFP reporter lines, which showed 98.9% concordance in organoid models .

How can NRL antibodies be used in genome editing validation studies?

NRL antibodies play a crucial role in validating CRISPR/Cas9-mediated genome editing outcomes:

• Editing efficiency assessment:

  • Western blot analysis can quantify reduction in NRL protein levels following knockout

  • Immunofluorescence can reveal spatial patterns of editing in heterogeneous tissues

• Phenotypic validation:

  • In Pde6a mouse models, anti-NRL antibodies confirmed successful Nrl gene inactivation

  • This validation was critical to demonstrating rescue of retinal degeneration

• Off-target effect analysis:

  • Immunostaining for NRL in non-target tissues can help identify unexpected effects

  • Combined with deep sequencing for comprehensive validation

When validating SaCas9-mediated Nrl inactivation in mouse models, Western blot with anti-NRL antibody (Proteintech #17388-1-AP) provided critical evidence of successful editing at the protein level .

What insights can NRL antibodies provide about species differences in retinal development?

NRL antibodies have revealed important evolutionary insights across vertebrate species:

• Conservation and divergence:

  • Despite high sequence conservation (90-94% identity across mammals), functional differences exist

  • Avian lineages have lost detectable NRL orthologs and likely rely on MAFA for rod specification

  • Zebrafish Nrl shows conserved SUMOylation sites and produces similar doublet bands on Western blots

• Functional conservation testing:

  • NRL antibodies help confirm protein expression in cross-species studies

  • Antibodies validate transgenic expression in rescue experiments

  • Immunodetection reveals species-specific developmental timing differences

• Comparative studies:

  • Single-cell analyses combined with NRL immunostaining reveal evolutionarily conserved and species-specific mechanisms in retinal development

  • This approach has identified both conserved and divergent transcriptional networks across species

Research using NRL antibodies has helped establish that while NRL function is largely conserved in rod specification across vertebrates, subtle species differences exist in developmental timing and regulatory networks .