nrarpb Antibody

What is NRARP and why is it important in cancer research?

NRARP (Notch-Regulated Ankyrin Repeat Protein) is a 13 kDa protein that functions as a negative feedback regulator in the Notch signaling pathway. It contains two C-terminal ankyrin-like repeats (amino acids 50-82 and 83-114) involved in protein-protein interactions . NRARP is critically important in cancer research because:

• It has been implicated in multiple malignancies including non-small cell lung cancer (NSCLC), T-cell acute lymphoblastic leukemia (T-ALL), colorectal, thyroid, breast, and liver cancers

• It exhibits dual regulatory functions, interacting with both Notch and Wnt signaling pathways

• High NRARP expression correlates with poor prognosis in certain cancers, making it a potential biomarker

• NRARP may represent a novel therapeutic target, particularly in cancers with aberrant Notch signaling

What are the main applications of NRARP antibodies in research?

NRARP antibodies are utilized across multiple experimental techniques:

Researchers should prioritize antibodies validated for their specific application of interest, as performance can vary significantly between techniques .

How should NRARP antibodies be optimized for Western blotting experiments?

For optimal Western blotting with NRARP antibodies:

• Sample preparation: NRARP migrates at approximately 25-30 kDa on SDS-PAGE despite its predicted molecular weight of 13 kDa . Use reducing conditions and appropriate buffer systems (e.g., Immunoblot Buffer Group 8 has been validated for some antibodies) .

• Antibody selection: Choose antibodies recognizing specific epitopes relevant to your research question. Available options include:

  • Antibodies recognizing amino acids 41-114

  • Antibodies targeting the full-length protein (amino acids 1-114)

  • Antibodies specific to N-terminal or middle regions

• Dilution optimization: Begin with manufacturer-recommended dilutions (typically 1:500-1:2,000) and optimize as needed .

• Controls: Include positive controls such as human placenta, liver, or kidney tissue lysates, which have demonstrated NRARP expression .

• Detection systems: Use appropriate secondary antibodies (e.g., HRP-conjugated anti-rabbit IgG for rabbit polyclonal primaries) .

What considerations are important when designing immunohistochemistry experiments with NRARP antibodies?

When conducting IHC studies with NRARP antibodies:

• Tissue processing: Both frozen and paraffin-embedded sections can be used, but protocol optimization differs. For paraffin sections, appropriate antigen retrieval is critical .

• Antibody selection: Choose antibodies validated specifically for IHC applications. Not all Western blot-validated antibodies perform well in IHC .

• Controls: Include:

  • Positive controls: Breast cancer tissues have been validated as positive controls due to their high NRARP expression

  • Negative controls: Replace primary antibody with PBS in serial sections

• Visualization: NRARP staining is predominantly cytoplasmic in tumor cells .

• Scoring systems: For quantitative analysis, consider using established scoring systems such as the immunoreactive score (IRS) system described in clinical studies .

How can NRARP antibodies be used to investigate the dual role of NRARP in cancer?

NRARP exhibits context-dependent functions that require sophisticated experimental approaches:

• T-ALL model systems: In T-cell acute lymphoblastic leukemia, NRARP can have either pro- or anti-tumoral effects depending on Notch activation status:

  • In cells with high NOTCH1 activity: NRARP overexpression blocks NOTCH signaling and delays proliferation

  • In cells with lower NOTCH1 activity: NRARP promotes cell expansion

Research protocol design should include:

• Antibodies that detect endogenous NRARP in conjunction with NOTCH1 activation markers

• Stratification of samples based on NOTCH1 mutational status and NICD1 levels

• Parallel assessment of both Notch and Wnt pathway activation

• Combination of NRARP detection with functional assays (proliferation, viability)

• NSCLC investigations: For non-small cell lung cancer studies:

  • Compare NRARP expression between tumor and adjacent normal tissues

  • Correlate with clinicopathological features (differentiation, TNM stage, smoking status)

  • Assess relationship with patient survival data

What experimental approaches can elucidate NRARP's interaction with the Wnt signaling pathway?

To investigate NRARP-Wnt pathway interactions:

• Co-immunoprecipitation studies: Use NRARP antibodies to pull down protein complexes and analyze interactions with Wnt pathway components, particularly LEF1 (lymphoid enhancer-binding factor 1) .

• Sequential detection protocol:

  • Use NRARP antibodies for initial detection

  • Strip and reprobe membranes with antibodies against LEF1 and other Wnt pathway components

  • Confirm interactions through reciprocal co-IP experiments

• Pathway activation analysis: Combine NRARP antibody detection with assessment of Wnt target gene expression, particularly in contexts with varying levels of Notch signaling .

• Recombinant protein studies: Exposure of primary cells to recombinant NRARP protein can help validate pathway interactions, as demonstrated in T-ALL PDX models .

How do I validate NRARP antibody specificity to ensure reliable results?

Comprehensive validation should include:

• Molecular weight verification: Confirm detection at the expected molecular weight (~25-30 kDa on SDS-PAGE despite 13 kDa predicted size) .

• Multiple antibody approach: Use antibodies targeting different epitopes of NRARP:

  • N-terminal region antibodies

  • Middle region antibodies

  • C-terminal/ankyrin repeat region antibodies

• Genetic manipulation controls:

  • NRARP-overexpressing cells as positive controls

  • NRARP knockdown/knockout cells as negative controls

• Cross-species reactivity assessment: Human and mouse NRARP are identical in amino acid sequence, allowing for cross-validation in multiple model systems .

• Tissue expression pattern verification: Compare expression patterns with published literature to ensure consistency with known NRARP distribution .

How should I interpret contradictory NRARP expression data in cancer studies?

When facing contradictory NRARP expression data:

• Context consideration: Remember that NRARP functions depend heavily on cellular context, particularly Notch pathway activation status .

• Methodological differences analysis:

  • Compare antibody epitopes used in different studies

  • Assess differences in detection methods (IHC vs. WB vs. mRNA)

  • Evaluate scoring systems and quantification approaches

• Patient stratification review: Check if patient populations were stratified by relevant factors:

  • For NSCLC: differentiation, TNM stage, smoking status

  • For T-ALL: NOTCH1 mutational status and NICD1 levels

• Pathway interaction analysis: Consider assessing both Notch and Wnt pathway activation simultaneously, as NRARP's effects depend on the balance between these pathways .

How can NRARP antibodies be utilized in developing potential cancer biomarkers?

• Tissue microarray analysis: Use validated NRARP antibodies to screen large patient cohorts with standardized IHC protocols.

• Multiplexed detection systems: Combine NRARP antibodies with other cancer markers to develop comprehensive prognostic panels.

• Validation methodology:

  • Compare NRARP protein detection with mRNA expression

  • Correlate with established prognostic indicators

  • Perform multivariate analysis to establish independent prognostic value

• Predictive biomarker development: Investigate correlation between NRARP expression and response to Notch-targeting therapies.

What are the considerations for using NRARP antibodies in therapeutic target validation studies?

When evaluating NRARP as a potential therapeutic target:

• Selective inhibition studies: Use NRARP antibodies to monitor protein levels after treatment with candidate inhibitors.

• Dual pathway consideration: Design experiments that account for NRARP's roles in both Notch and Wnt pathways:

  • In high Notch contexts, inhibiting NRARP might be detrimental (enhancing Notch signaling)

  • In low Notch contexts, inhibiting NRARP might be beneficial (reducing Wnt signaling)

• Patient stratification markers: Develop companion diagnostic approaches using NRARP antibodies to identify patients likely to respond to pathway-specific therapies.

• Therapeutic resistance monitoring: Investigate NRARP expression changes during treatment and potential correlation with resistance development.