NR2C2AP Antibody

What is NR2C2AP and what functional role does it play in cellular processes?

NR2C2AP (Nuclear Receptor 2C2-Associated Protein, also known as TRA16) is a 16 kDa protein primarily localized in the nucleus that functions as a transcriptional regulator. This protein acts as a repressor of NR2C2-mediated transactivation by suppressing the binding between NR2C2/TR4 and the TR4-response element in target genes . NR2C2AP exerts its repressive function by blocking the interaction between TR4 and TR4 ligand-binding domain through direct interaction with TR4-DNA-binding and ligand-binding domains . The protein is encoded by a gene located on chromosome 19p13.11 and contains 6 exons . Understanding NR2C2AP's role in gene regulation provides critical context for experimental design using antibodies against this target.

What types of NR2C2AP antibodies are available for research applications?

Current research-grade NR2C2AP antibodies are predominantly polyclonal antibodies raised in rabbits. These antibodies react with human, mouse, and rat samples . Available formats include:

Most commercially available antibodies target either the full-length protein or specific epitopes, with some generated against recombinant fusion proteins containing sequences corresponding to amino acids 1-139 of human NR2C2AP . For optimal results, researchers should select antibodies validated specifically for their experimental system and application.

How should I optimize Western blot protocols when using NR2C2AP antibodies?

Successful Western blot experiments with NR2C2AP antibodies require careful optimization:

When troubleshooting, note that non-specific bands may appear due to cross-reactivity with other nuclear proteins. Extensive blocking (5% non-fat milk or BSA for 1-2 hours) and thorough washing steps can minimize background signal.

What are the recommended protocols for validating the specificity of an NR2C2AP antibody?

A robust validation strategy for NR2C2AP antibodies should include:

• Positive and negative control tissues/cells: Compare expression in tissues known to express NR2C2AP versus those with minimal expression.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to confirm signal specificity.

• Knockdown/knockout validation: Compare antibody reactivity in wild-type versus NR2C2AP knockdown/knockout samples.

• Multiple antibody comparison: Test multiple antibodies targeting different epitopes of NR2C2AP to confirm consistent patterns.

• Cross-species reactivity testing: Validate antibody performance across human, mouse, and rat samples if cross-species applications are planned .

The gold standard for antibody validation includes orthogonal approaches such as correlating protein detection with mRNA expression data and using recombinant expression systems as positive controls, as implemented in enhanced validation protocols .

Why might I observe inconsistent results when using NR2C2AP antibodies in immunohistochemistry?

Inconsistent immunohistochemistry results with NR2C2AP antibodies may stem from several factors:

• Fixation variables: NR2C2AP epitopes may be sensitive to overfixation. Optimize fixation time (typically 24-48 hours in 10% neutral buffered formalin) and consider comparing multiple fixatives.

• Antigen retrieval methods: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is generally recommended, but some epitopes may require alternative buffers like EDTA (pH 9.0).

• Antibody concentration: Most NR2C2AP antibodies require dilutions between 1:20-1:200 for IHC applications . Titrate the antibody to determine optimal concentration for your specific tissue.

• Detection system sensitivity: Consider using amplification systems (e.g., tyramide signal amplification) for low-abundance targets.

• Nuclear staining specificity: Since NR2C2AP is a nuclear protein, confirm proper nuclear localization of the signal. Non-nuclear staining may indicate non-specific binding.

When optimizing, include positive control tissues with known NR2C2AP expression and negative controls (primary antibody omission and isotype controls) in each experiment.

How can I interpret changes in NR2C2AP localization or expression patterns in different experimental conditions?

Interpreting NR2C2AP localization requires understanding its normal distribution and potential changes:

• Normal localization: NR2C2AP is predominantly localized in the nucleoplasm . Nuclear speckles or nucleolar exclusion may be visible at high magnification.

• Quantification approaches: For expression changes, consider:

  • Western blot densitometry normalized to nuclear loading controls (e.g., Lamin B)

  • Immunofluorescence intensity measurements with nuclear counterstains

  • Cell counting for percent positive nuclei in tissue sections

• Co-localization studies: Co-stain with NR2C2/TR4 to assess functional relationships, as NR2C2AP represses NR2C2-mediated transactivation .

• Dynamic studies: In cell culture models, NR2C2AP localization may change during cell cycle or in response to signaling pathways affecting nuclear receptor activity.

When interpreting changes in experimental conditions, consider whether alterations represent changes in protein abundance, localization, or post-translational modifications that might affect epitope recognition.

How can NR2C2AP antibodies be used to investigate protein-protein interactions with the TR4/NR2C2 pathway?

NR2C2AP antibodies provide powerful tools for studying protein-protein interactions in the TR4/NR2C2 pathway:

• Co-immunoprecipitation (Co-IP): Use NR2C2AP antibodies for immunoprecipitation followed by Western blot detection of interacting partners. The antibody's ability to recognize native protein conformation is critical for this application.

• Proximity Ligation Assay (PLA): Combine NR2C2AP antibodies with antibodies against potential interacting partners to visualize and quantify protein interactions with subcellular resolution.

• Chromatin Immunoprecipitation (ChIP): NR2C2AP functions as a repressor of NR2C2-mediated transactivation , making ChIP a valuable approach to study its role in transcriptional regulation. Use NR2C2AP antibodies to:

  • Identify genomic regions bound by NR2C2AP

  • Assess co-occupancy with NR2C2/TR4 at target promoters

  • Study recruitment of additional co-repressors

• Sequential ChIP (Re-ChIP): Determine if NR2C2AP and NR2C2/TR4 simultaneously occupy the same genomic regions using sequential immunoprecipitation with both antibodies.

When designing these experiments, consider that NR2C2AP suppresses binding between NR2C2/TR4 and TR4-response elements , suggesting competitive or inhibitory interactions that may be context-dependent.

What approaches can be used to study the effects of anti-NR2 antibodies on blood-brain barrier models as demonstrated in neuropsychiatric lupus research?

Studies examining anti-NR2 antibodies' effects on blood-brain barrier (BBB) models employ systematic approaches that could inform NR2C2AP research:

• In vitro BBB model construction:

  • Isolate and culture primary brain microvessel endothelial cells

  • Establish BBB models measuring transepithelial electrical resistance (TEER)

  • Apply treatments including antibodies, agonists, and antagonists

• Permeability assessment protocols:

  • Measure TEER values following antibody treatment

  • Compare effects across multiple conditions (positive/negative controls)

  • Determine time-dependent changes in barrier integrity

• Experimental design structure: Organize experiments in multiple groups:

  • Group A: Testing anti-NR2 antibody, positive/negative CSF, positive/negative serum

  • Group B: Testing interactions between anti-NR2 antibody and receptor modulators

  • Group C: Examining protective effects of compounds like dexamethasone

In one study, TEER values decreased by 54.6% after anti-NR2 antibody addition compared to controls, suggesting increased BBB permeability . This methodological framework could be adapted to investigate whether NR2C2AP antibodies affect similar cellular barriers or to study NR2C2AP's role in maintaining cellular junctions.

How can NR2C2AP antibodies be used in tissue microarray analysis to establish expression patterns across multiple cancer types?

NR2C2AP antibodies can be valuable tools for comprehensive tissue microarray (TMA) analysis in cancer research:

• High-throughput screening protocol:

  • Optimize antibody concentration for TMA sections (typically 1:50-1:200 dilution)

  • Include positive and negative control tissues on each TMA

  • Use automated staining platforms for consistency across multiple slides

• Scoring and quantification approaches:

  • Develop clear scoring criteria for nuclear NR2C2AP expression (intensity, percent positive)

  • Employ digital pathology and image analysis software for unbiased quantification

  • Correlate expression patterns with clinical parameters and molecular subtypes

• Validation through orthogonal methods:

  • Confirm key findings with alternative antibodies targeting different epitopes

  • Validate expression at mRNA level through in situ hybridization or transcriptomic data

  • Perform functional studies in cell lines representing identified high/low expression groups

The Prestige Antibodies® program has established protocols for TMA analysis of multiple normal human tissues (44) and common cancer types (20) , providing a framework for comprehensive NR2C2AP expression mapping across tissue types.

What are the optimal storage conditions for maintaining NR2C2AP antibody activity during long-term research projects?

Proper storage is critical for maintaining NR2C2AP antibody activity throughout extended research projects:

• Temperature requirements:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

  • For short-term use (days), storage at 4°C is acceptable for some formulations

• Buffer composition impact:

  • Most NR2C2AP antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

  • Some formulations include stabilizers like 0.1% BSA or 2% sucrose

  • Avoid diluting stock antibody unless immediately using

• Stability considerations:

  • Properly stored antibodies remain stable for approximately one year after shipment

  • Monitor performance periodically using positive controls

  • Document lot-to-lot variations if using antibodies over extended periods

For critical applications, consider setting aside reference aliquots from well-performing lots to validate new antibody batches, ensuring experimental continuity across long-term studies.

What methodological approaches can enhance reproducibility when working with NR2C2AP antibodies across different experimental batches?

Ensuring reproducibility with NR2C2AP antibodies requires systematic approaches:

• Antibody validation documentation:

  • Maintain detailed records of antibody source, catalog number, lot number, and validation results

  • Document optimal working conditions for each application (dilution, incubation time/temperature)

  • Create laboratory SOPs for key applications

• Standardized protocols:

  • Use consistent sample preparation methods

  • Standardize blocking solutions and washing procedures

  • Implement positive and negative controls in every experiment

• Quantitative benchmarking:

  • Establish expected signal intensity ranges for standard samples

  • Use calibrated protein standards for Western blot

  • Include reference cell lines with known NR2C2AP expression levels

When changing antibody lots, perform side-by-side comparison experiments to assess potential differences in specificity, sensitivity, or background. This comparative data enables appropriate adjustments to maintain consistent results across your research program.