NR6A1 Antibody

What is NR6A1 and why are antibodies against it important for research?

NR6A1 (Nuclear Receptor Subfamily 6 Group A Member 1) is a nuclear receptor protein involved in transcription regulation. In humans, the canonical protein has 480 amino acid residues with a molecular mass of 54.4 kDa and is localized in the nucleus. NR6A1 is also known by several synonyms including GCNF, GCNF1, RTR, hGCNF, and retinoic acid receptor-related testis-associated receptor .

Antibodies against NR6A1 are crucial for multiple research applications because:

• They enable detection and quantification of NR6A1 in various experimental systems

• They allow visualization of subcellular localization patterns

• They facilitate investigations into NR6A1's role in transcriptional regulation

• They support studies of NR6A1's interactions with target DNA sequences

• They help elucidate NR6A1's involvement in developmental processes and disease mechanisms

What are the principal applications of NR6A1 antibodies in molecular biology research?

NR6A1 antibodies support multiple experimental applications with specific methodological considerations:

For Western blot applications, different antibodies target specific regions of NR6A1, with many recognizing the ligand binding domain or specific amino acid sequences (e.g., AA 65-118) .

How should researchers optimize ChIP-PCR protocols when using NR6A1 antibodies?

Chromatin Immunoprecipitation (ChIP) with NR6A1 antibodies requires specific methodological considerations:

• Cross-linking optimization:

  • Use 1% formaldehyde for cross-linking (10-15 minutes at room temperature)

  • Quench with 0.125M glycine

  • Prepare nuclear extract according to established protocols

• DNA fragmentation:

  • Digest genomic DNA using Micrococcal Nuclease to obtain fragments ranging from 150-900 bp

  • Confirm fragment sizes via gel electrophoresis

• Immunoprecipitation:

  • Use 10μg of anti-GCNF monoclonal antibody for optimal capture of NR6A1 protein

  • Incubate with Protein-G agarose beads

  • Wash NR6A1-antibody-Protein-G complexes thoroughly

• PCR amplification:

  • Design primers targeting putative NR6A1-binding sites (particularly DR0 elements and NurRE motifs)

  • Include controls with normal rabbit serum and water without DNA template

• Validation strategy:

  • Quantitative RT-PCR analysis in cells transfected with NR6A1 expression vectors to confirm increased binding to target regions

Note that the binding capacity of NR6A1 to specific DNA motifs like NurRE may be relatively low, requiring optimization of antibody concentration and incubation conditions .

What approaches are recommended for visualizing NR6A1 expression in tissue sections?

For effective immunohistochemical detection of NR6A1 in tissue sections, researchers should follow these methodological steps:

• Tissue preparation:

  • For paraffin sections: Fix tissue in 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4)

  • Post-fix appropriately before cryosectioning into 30-μm sections

  • For formalin-fixed paraffin-embedded tissues, prepare 4-μm sections on pre-cleaned charged microscope slides

• Antigen retrieval:

  • Heat slides in a tissue-drying oven (45 minutes at 60°C)

  • Deparaffinize with three 5-minute washes in xylene at room temperature

• Double immunohistochemical staining protocol:

  • Incubate sections with rabbit anti-HCRT-1 antiserum (1:5000) and mouse anti-GCNF monoclonal antibody (1:500)

  • Use secondary antibodies such as goat Alexa594-labeled anti-mouse IgG and goat Alexa488-labeled anti-rabbit IgG (1:2000)

  • Visualize under a fluorescence microscope equipped with a digital camera

• Specificity controls:

  • Include negative controls (e.g., MOLT-4 human acute lymphoblastic leukemia cell line)

  • Include positive controls (e.g., HepG2 human hepatocellular carcinoma cell line)

  • Counter-stain nuclei with DAPI to confirm nuclear localization

The recommended antibody concentration for immunohistochemistry is typically 4-5 μg/mL .

How can researchers evaluate DNA binding properties of NR6A1 using antibodies?

To study NR6A1's interaction with DNA, researchers can implement these methodological approaches:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Prepare radiolabeled oligonucleotide probes containing direct repeat with zero spacing (DR0) of the consensus sequence AGGTCA

  • Incubate with native or mutated NR6A1 proteins

  • Verify binding specificity through competition assays using unlabeled DR0 sequences

  • Confirm NR6A1 presence in the complex via supershift assays with anti-tag antibodies (e.g., anti-Myc)

• Competitive inhibition assays:

  • Introduce wildtype and mutated DR0 site DNA in increasing amounts

  • Observe diminished binding with increasing wildtype DNA compared to mutated sequences

  • This confirms selective binding to specific DNA motifs

• Targeted ChIP assays:

  • Differentiate mouse embryonic stem cells (mESC) into neural crest cells (NCC)

  • Evaluate binding to DR0 sites in genes of interest (e.g., Sox9, Sox10, Snai1, Zeb2)

  • Also assess binding to other known targets like Nanog DR0 sites

These approaches can reveal how variants in NR6A1 affect DNA binding affinity, with implications for understanding developmental disorders linked to NR6A1 mutations .

What methodologies are effective for studying NR6A1's role in transcriptional regulation?

To investigate NR6A1's function in regulating gene expression, researchers should consider:

• Chromatin accessibility analysis:

  • Compare chromatin accessibility of target genes (e.g., Oct4) in NR6A1-/- mutant embryos versus controls

  • Identify peaks showing increased or decreased accessibility of open chromatin

  • Align these with locations of putative DR0 binding sites

• Reporter gene assays:

  • Construct reporter plasmids containing promoter regions with NR6A1 binding sites

  • Co-transfect with NR6A1 expression vectors (wildtype or mutant)

  • Measure transcriptional activity changes to determine repressive or activating functions

• Gene expression analysis after NR6A1 manipulation:

  • Perform temporal deletion studies using conditional knockout models

  • Analyze expression of target genes using RT-qPCR

  • Evaluate changes in regulatory networks (e.g., pluripotency factors, neural crest specifiers)

• In vivo overexpression studies:

  • Design temporal initiation of Oct4 overexpression to correlate with the temporal requirement for Nr6a1

  • Assess disruption of neural crest cell specification and formation

These approaches have revealed NR6A1's dual role as a repressor of pluripotency factors and an activator of gene regulatory networks involved in neural crest cell specification .

What are common challenges with NR6A1 antibodies and how can they be addressed?

When troubleshooting, remember that the binding capacity of NR6A1 to certain DNA motifs like NurRE may be naturally low , which might necessitate optimizing experimental conditions or using more sensitive detection methods.

How should researchers select the most appropriate NR6A1 antibody for specific applications?

When selecting NR6A1 antibodies, consider these methodological factors:

• Target epitope considerations:

  • Ligand Binding Domain antibodies: Suitable for studying NR6A1's function in transcriptional regulation

  • N-terminal region antibodies (AA 30-59): Useful for detecting full-length protein

  • Mid-region antibodies: Can detect most isoforms and fragments

• Application-specific selection:

  • For Western blot: Both monoclonal and polyclonal antibodies work well; consider epitope accessibility after denaturation

  • For ChIP: High-affinity antibodies recognizing native conformation are essential

  • For immunohistochemistry: Polyclonal antibodies often provide stronger signals in tissues

• Species cross-reactivity needs:

  • Wide cross-reactivity (human, mouse, rat, etc.): Some antibodies show reactivity across species (up to 100% sequence identity)

  • Species-specific detection: Choose antibodies validated for specific species

• Validation status:

  • Prioritize antibodies with published citations

  • Consider antibodies validated by multiple techniques (WB, IHC, IF)

  • Check if specificity has been confirmed by knockout/knockdown controls

For ChIP applications specifically, monoclonal antibodies like anti-GCNF (Perseus Proteomics, PP-H7921-00) have been successfully used in published research .

How are NR6A1 antibodies being used to investigate developmental biology and disease mechanisms?

Recent research has employed NR6A1 antibodies in several cutting-edge applications:

• Neural crest cell (NCC) development studies:

  • NR6A1 antibodies help visualize expression patterns during embryonic development

  • They reveal NR6A1's role in repressing pluripotency factors while activating NCC specification genes

  • Immunohistochemistry with NR6A1 antibodies shows temporal-spatial expression patterns critical for understanding developmental timing

• Investigation of congenital disorders:

  • NR6A1 variants have been linked to renal, uterine, and costovertebral defects

  • Antibodies enable detection of mutant NR6A1 proteins with decreased DNA binding affinity

  • They help characterize how heterozygous loss-of-function variants affect development

• TGF-β signaling pathway research:

  • Studies of lnc-Nr6a1 as a reservoir of miR during epithelial-mesenchymal transition (EMT)

  • Expression levels of lnc-Nr6a1 isoforms and SIP1 measured by RT-qPCR analysis

  • Antibodies help track the regulation and function of NR6A1 in these processes

• Transcriptional regulation of hypocretin/orexin:

  • NR6A1 antibodies in ChIP-PCR studies demonstrated binding to the prepro-hypocretin promoter

  • Double immunohistochemical staining revealed co-expression patterns

  • These studies have implications for understanding sleep regulation mechanisms

What advanced techniques combine NR6A1 antibodies with other methodologies for deeper mechanistic insights?

Researchers are integrating NR6A1 antibodies into sophisticated multi-modal approaches:

• Multiomics integration:

  • Combining ChIP-seq with ATAC-seq to correlate NR6A1 binding with chromatin accessibility changes

  • Integrating RNA-seq data to connect binding events with transcriptional outcomes

  • These approaches reveal how NR6A1 modulates the chromatin landscape during development

• iDRIP-MS (Identification of Direct RNA-Interacting Proteins by Mass Spectrometry):

  • Reveals direct lnc-Nr6a1-1-interacting proteins

  • Complements antibody-based studies by identifying protein interaction networks

  • Efficiency of lnc-Nr6a1-1 pulldown verified by RT-qPCR against standard curves

• Combined in vivo and in vitro approaches:

  • Using human induced pluripotent stem cells (iPSCs) differentiated into NCCs alongside mouse models

  • Antibodies facilitate tracking of NR6A1 expression across different experimental systems

  • This enables translation between model systems and human development

• Conditional spatiotemporal deletion models:

  • Global temporal and conditional spatiotemporal deletion reveals specific requirements for NR6A1

  • Antibodies confirm deletion efficiency and help track developmental consequences

  • These approaches have revealed that NCC specification in mouse embryos commences earlier than previously recognized

By employing these integrated approaches, researchers are uncovering NR6A1's complex roles in development and disease, establishing it as a novel regulator of mammalian NCC specification and formation .