NGFRAP1 Antibody

What is NGFRAP1 and why is it important for molecular research?

NGFRAP1 (nerve growth factor receptor associated protein 1) is a 24 kDa member of the BEX protein family that contains a nuclear export signal (amino acids 77-87) allowing it to export bound p75NTR. It functions primarily as a p75NTR-associated death executor (NADE), mediating apoptosis through interaction with the cell death domain of p75NTR following nerve growth factor binding. NGFRAP1 is widely expressed throughout the body, with highest expression in ovarian granulosa cells, testis, prostate, seminal vesicle, and liver .

Research on NGFRAP1 has expanded beyond its initial characterization as an ovarian granulosa cell protein to reveal significant functions in neurotrophin signaling, apoptotic regulation, and potential roles in disease pathogenesis. Recent studies have implicated NGFRAP1/BEX3 in cancer chemoresistance and interactions with second mitochondria-derived activator of caspase (Smac), highlighting its importance as a research target for understanding complex cellular death mechanisms .

What are the key aliases and nomenclature for NGFRAP1 antibodies?

NGFRAP1 is known by several aliases in scientific literature, which is important to recognize when searching for relevant antibodies and publications:

Understanding these various nomenclatures is essential when designing experimental strategies and conducting literature searches, as different research groups may use different terms to refer to the same protein .

What molecular weight should I expect for NGFRAP1 in Western blot analyses?

When working with NGFRAP1 antibodies in Western blot applications, researchers should be aware of the discrepancy between calculated and observed molecular weights:

This consistent migration at higher-than-predicted molecular weights across multiple studies suggests potential post-translational modifications or structural features affecting electrophoretic mobility. Researchers should consider these variations when interpreting Western blot results and not automatically dismiss bands appearing at these higher molecular weights as non-specific .

How does NGFRAP1/BEX3 contribute to apoptotic signaling pathways?

NGFRAP1/BEX3 operates as a multifunctional regulator in apoptotic signaling through several distinct mechanisms:

First, as a p75NTR-associated death executor (NADE), it mediates neurotrophin-induced apoptosis by interacting directly with the cell death domain of p75NTR following NGF binding. This interaction initiates downstream death signaling cascades that ultimately lead to cellular apoptosis .

Second, NGFRAP1/NADE forms critical interactions with Smac (second mitochondria-derived activator of caspase), which significantly enhances TRAIL-induced apoptosis. This interaction serves a dual purpose: NADE not only promotes Smac's pro-apoptotic functions but also inhibits XIAP-mediated Smac ubiquitization, thereby preventing its degradation and maintaining its antagonistic effect on apoptosis inhibitors .

The relationship between NGFRAP1 and apoptotic regulation makes it a potential therapeutic target in diseases characterized by dysregulated cell death, including neurodegenerative disorders and certain cancers. Understanding the molecular details of these interactions provides valuable insights into fundamental cell death mechanisms and potential intervention strategies .

What is the emerging role of NGFRAP1/BEX3 in cancer chemoresistance?

Recent research has revealed a significant role for NGFRAP1/BEX3 in modulating cancer cell responses to chemotherapeutic agents. A 2017 study by Gao et al. specifically investigated how BEX3 contributes to cisplatin chemoresistance in nasopharyngeal carcinoma .

The researchers utilized comprehensive experimental approaches including immunocytochemistry and immunohistochemistry on both human and mouse models to characterize this relationship. Their findings indicated that BEX3 expression correlates with reduced sensitivity to cisplatin treatment in nasopharyngeal carcinoma cells, suggesting its potential as a biomarker for treatment resistance .

This chemoresistance mechanism likely intersects with NGFRAP1's known functions in apoptotic regulation. Since many chemotherapeutic agents, including cisplatin, induce cell death through apoptotic pathways, NGFRAP1's ability to modulate these pathways could directly impact treatment efficacy. By potentially altering the threshold for apoptotic activation, NGFRAP1/BEX3 may allow cancer cells to evade drug-induced cell death .

These findings suggest that targeting NGFRAP1/BEX3 could be a valuable strategy for overcoming chemoresistance in certain cancers, particularly those dependent on altered apoptotic thresholds for survival and drug resistance.

How do interactions between NGFRAP1/NADE and Smac influence the apoptotic threshold?

The interaction between NGFRAP1/NADE and Smac represents a critical regulatory node in apoptotic signaling that significantly influences cellular sensitivity to death signals. This molecular partnership functions through multiple mechanisms to promote effective apoptosis:

First, co-expression studies have demonstrated that NADE and Smac together substantially enhance TRAIL-induced apoptosis, suggesting synergistic amplification of death receptor signaling .

Second, NADE directly counteracts anti-apoptotic defenses by inhibiting XIAP-mediated ubiquitization of Smac. This biochemical intervention is critically important because:

• XIAP (X-linked inhibitor of apoptosis protein) normally suppresses apoptosis by inhibiting caspase activation

• Smac naturally antagonizes XIAP, promoting apoptosis progression

• XIAP targets Smac for proteasomal degradation through ubiquitization

• NADE's prevention of this ubiquitization preserves Smac's pro-apoptotic functions

This molecular arrangement creates a regulatory circuit where NGFRAP1/NADE can effectively lower the threshold for apoptotic induction by preserving and enhancing Smac's antagonism of XIAP. This mechanism provides valuable insight into how cells fine-tune their sensitivity to apoptotic stimuli and suggests potential targets for therapeutic intervention in diseases characterized by apoptotic dysregulation .

What are the optimal conditions for Western blot analysis using NGFRAP1 antibodies?

For reliable Western blot detection of NGFRAP1/BEX3, researchers should follow these optimized protocols based on validated antibody performance:

Key technical considerations:

• Include appropriate positive controls (HT-29 cells, mouse liver) to confirm detection

• Be aware of species differences in NGFRAP1 structure (rodent-specific Arg/His region at aa 35)

• The discrepancy between calculated and observed molecular weights is normal for this protein

• For problematic detection, extended exposure times or enhanced chemiluminescence may improve results

What protocol should be followed for immunohistochemical detection of NGFRAP1?

For optimal immunohistochemical detection of NGFRAP1/BEX3 in tissue sections, follow this validated protocol:

Sample Preparation:

• Use immersion-fixed, paraffin-embedded tissue sections (validated with human prostate and stomach cancer tissues)

• Section tissues at 4-6 μm thickness and mount on positively charged slides

Antigen Retrieval (Critical Step):

• Perform heat-induced epitope retrieval using one of these validated methods:

  • Antigen Retrieval Reagent-Basic (R&D Systems, CTS013)

  • TE buffer pH 9.0 (recommended for Proteintech antibody)

  • Alternative: citrate buffer pH 6.0

• Heat slides to 95-100°C for 15-20 minutes in retrieval solution

• Allow slides to cool gradually to room temperature in retrieval solution

Antibody Incubation:

• For R&D Systems MAB6858: Use at 15 μg/mL, incubate overnight at 4°C

• For Proteintech 10446-1-AP: Use at 1:250-1:1000 dilution

• For Boster Bio NADE antibody: Start at 2 μg/mL

Detection and Visualization:

• For mouse primary antibodies: Use Anti-Mouse HRP-DAB detection system (e.g., R&D Systems CTS002)

• Counterstain with hematoxylin for nuclear visualization

• In human prostate tissue, expect specific staining localized to epithelial cells

Quality Control Measures:

• Include positive control tissues (human prostate, stomach cancer)

• Run parallel negative controls (primary antibody omitted)

• Consider peptide competition controls to confirm specificity

This protocol has been validated to produce specific staining of NGFRAP1/BEX3 in human tissues with minimal background and high signal-to-noise ratio.

How should researchers validate the specificity of NGFRAP1 antibodies?

Rigorous validation of NGFRAP1 antibody specificity is essential for generating reliable and reproducible research data. Implement these validation strategies:

1. Western Blot Validation:

• Verify detection of a single band at expected molecular weight (15-28 kDa, depending on antibody)

• Test across multiple expressing cell lines (HT-29, Balb/3T3, C2C12, HepG2)

• Compare observed band patterns with manufacturer's reference data

2. Peptide Competition Assay:

• Perform parallel Western blots or IHC with antibody pre-incubated with blocking peptide

• Specific signals should disappear or significantly diminish with blocking peptide

• Boster Bio documents this approach for their NADE antibody in human brain lysates

3. Cross-Species Reactivity Testing:

• Test antibody across multiple species (human, mouse, rat)

• Verify reactivity patterns match expected homology (human NGFRAP1 shares 94% aa identity with mouse/rat)

• Note species-specific features (13-19 aa rodent Arg/His region at aa 35 not found in humans)

4. Immunohistochemistry Controls:

• Test in tissues with documented expression (prostate epithelial cells)

• Include appropriate negative controls

• Verify staining pattern matches known cellular localization

5. Direct ELISA Validation:

• R&D Systems antibody MAB6858 has been validated to detect human NGFRAP1/BEX3 in direct ELISAs

• This provides additional confirmation of binding specificity

Implementing these validation strategies will ensure confidence in the specificity of NGFRAP1 antibodies and the reliability of experimental results across different applications.

What approach should be taken when unexpected molecular weight bands appear in NGFRAP1 Western blots?

When encountering unexpected molecular weight bands in NGFRAP1 Western blots, researchers should follow this systematic troubleshooting approach:

Step 1: Determine if the unexpected bands are normal for NGFRAP1
NGFRAP1/BEX3 shows documented variation in migration patterns across different detection systems:

• Despite calculated MW of ~13 kDa, it commonly appears at 15-28 kDa

• R&D Systems reports detection at ~15 kDa in human and mouse cell lines

• Boster Bio notes migration at ~23 kDa "despite its predicted molecular weight"

• Proteintech reports an observed MW of 28 kDa

• Multiple isoforms exist: Proteintech lists calculated MWs of 13, 22, and 44 kDa

Step 2: Evaluate sample preparation factors

• Confirm use of reducing conditions (NGFRAP1 antibodies are validated under reducing conditions)

• Verify buffer composition matches recommended systems (e.g., "Immunoblot Buffer Group 1" for R&D Systems antibody)

• Check for potential protein degradation in samples

Step 3: Consider species-specific variations

• Rodent NGFRAP1 contains a 13-19 amino acid Arg/His region at aa 35 not found in humans

• This can affect migration patterns when comparing across species

Step 4: Validate with controls

• Run positive control lysates (e.g., HT-29, Balb/3T3 cells) alongside experimental samples

• Compare band patterns with manufacturer's reference data

• Consider peptide competition assays to identify specific bands

If unexpected bands persist after these steps, they may represent specific detection of NGFRAP1 variants, post-translationally modified forms, or protein complexes rather than non-specific binding.

How can researchers optimize antibody storage to maintain NGFRAP1 antibody activity?

Maintaining NGFRAP1 antibody activity requires careful attention to storage conditions. Follow these evidence-based guidelines for optimal antibody performance:

Long-term Storage Recommendations:

• Temperature: -20°C to -70°C under sterile conditions

• Duration: Up to 12 months from date of receipt (as supplied)

• For reconstituted antibodies: 6 months at -20°C to -70°C

• Use a manual defrost freezer to prevent temperature fluctuations during automated defrost cycles

Short-term Storage Options:

• Temperature: 2°C to 8°C (refrigerated)

• Duration: Up to 1 month under sterile conditions after reconstitution

• For example, Boster Bio's NADE antibody maintains stability at 4°C for three months

Reconstitution Guidelines:

• Lyophilized antibodies: Reconstitute in sterile PBS to 0.5 mg/mL final concentration

• Small pack size antibodies may be supplied as filtered solutions requiring no reconstitution

Critical Handling Practices:

• Aliquot antibodies into single-use volumes immediately after first thaw

• Avoid repeated freeze-thaw cycles which dramatically reduce activity

• Use sterile technique when handling antibody solutions

• Note that some formulations contain stabilizers:

  • R&D Systems: Lyophilized from PBS with Trehalose

  • Proteintech: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

  • Boster Bio: PBS containing 0.02% sodium azide

Following these storage recommendations will help maintain antibody activity and ensure reliable experimental results throughout the antibody's shelf life.

What controls are essential when performing NGFRAP1 immunohistochemistry?

When performing immunohistochemistry with NGFRAP1 antibodies, implementing proper controls is crucial for result interpretation and experimental validity. The following control panel should be included:

1. Positive Tissue Controls:

• Human prostate tissue (epithelial cells show specific NGFRAP1/BEX3 staining)

• Human stomach cancer tissue (validated for Proteintech antibody)

• These tissues serve as benchmarks for staining pattern and intensity

2. Negative Controls:

• Procedural negative control: Process sections identically but omit primary antibody

• Isotype control: Use irrelevant antibody of same isotype and concentration

  • For mouse monoclonal antibodies (R&D Systems): Mouse IgG2b

  • For rabbit polyclonal antibodies (Proteintech, Boster): Normal rabbit IgG

• These controls help identify non-specific binding of detection systems

3. Peptide Competition Control:

• Pre-incubate antibody with specific blocking peptide

• Process in parallel with standard antibody

• Specific staining should be eliminated or dramatically reduced

• Boster Bio demonstrates this approach in their validation data

4. Antigen Retrieval Controls:

• Include sections with and without antigen retrieval

• Compare different retrieval methods if optimizing protocol

  • Basic pH buffer (TE buffer pH 9.0, R&D Systems CTS013)

  • Acidic pH buffer (citrate buffer pH 6.0)

• This helps determine optimal epitope exposure conditions

5. Antibody Titration Controls:

• Test multiple antibody dilutions to determine optimal concentration

• R&D Systems: 8-25 μg/mL range recommended

• Proteintech: 1:250-1:1000 dilution range recommended

• This establishes optimal signal-to-noise ratio

Implementing this comprehensive control panel ensures valid interpretation of NGFRAP1 immunohistochemistry results and facilitates troubleshooting of any technical issues that may arise.

What approaches can resolve background issues in NGFRAP1 immunohistochemistry?

Background staining in NGFRAP1 immunohistochemistry can obscure specific signals and complicate data interpretation. Implement these evidence-based solutions to achieve optimal signal-to-noise ratio:

1. Optimize Antibody Concentration:

• Determine the minimum effective concentration through systematic titration:

  • R&D Systems MAB6858: Recommended range 8-25 μg/mL

  • Proteintech 10446-1-AP: Recommended range 1:250-1:1000

  • Boster Bio NADE antibody: Start at 2 μg/mL

• Lower concentrations often reduce non-specific binding while maintaining specific signals

2. Refine Antigen Retrieval Protocol:

• Select the optimal method for your specific antibody:

  • Basic pH: R&D Systems recommends Antigen Retrieval Reagent-Basic (CTS013)

  • Neutral/Basic pH: Proteintech suggests TE buffer pH 9.0

  • Acidic pH: Citrate buffer pH 6.0 is an alternative option

• Excessive retrieval can increase background; insufficient retrieval reduces specific signal

• Optimize retrieval time and temperature for your specific tissue

3. Implement Enhanced Blocking Strategies:

• Extend blocking time (30-60 minutes at room temperature)

• Use dual blocking approach:

  • Protein block: 2-5% BSA, 5-10% normal serum from secondary antibody species

  • Peroxidase block: 0.3-3% hydrogen peroxide (for HRP-based detection)

• Add 0.1-0.3% Triton X-100 to reduce non-specific hydrophobic interactions

4. Optimize Detection System:

• R&D Systems specifically recommends Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (CTS002)

• Fresh preparation of detection reagents improves performance

• Shorter DAB development times may reduce background while preserving specific staining

5. Enhance Washing Protocol:

• Increase wash buffer volume and number of wash steps

• Use gentle agitation during washes to improve removal of unbound antibodies

• Consider adding 0.05-0.1% Tween-20 to wash buffers to reduce non-specific binding

6. Sample-Specific Considerations:

• Different tissues may require customized protocols

• NGFRAP1/BEX3 shows specific staining in prostate epithelial cells

• Optimize based on target tissue characteristics and fixation conditions

By systematically implementing these strategies, researchers can achieve clean, specific staining for NGFRAP1/BEX3 in immunohistochemical applications with minimal background interference.

What emerging research directions are most promising for NGFRAP1 antibody applications?

NGFRAP1/BEX3 antibodies are becoming increasingly valuable tools for investigating several promising research directions:

First, cancer chemoresistance mechanisms represent a significant frontier. The 2017 study by Gao et al. demonstrating BEX3's contribution to cisplatin resistance in nasopharyngeal carcinoma suggests NGFRAP1 antibodies will be crucial for investigating resistance mechanisms across different cancer types. This could lead to the development of predictive biomarkers for treatment response and potential therapeutic targets for overcoming chemoresistance .

Second, the relationship between NGFRAP1/NADE and apoptotic regulation through Smac interaction offers promising research opportunities. Further characterization of how this interaction influences the sensitivity of cells to death signals could provide insights into fundamental cell death mechanisms and potential therapeutic strategies for diseases with dysregulated apoptosis .

Third, neurological applications merit exploration, particularly regarding NGFRAP1's suggested role in neuronal death during epileptic brain damage. Antibodies detecting different forms and modifications of NGFRAP1 could help elucidate its contributions to neurodegeneration and potentially identify neuroprotective strategies .

As research continues to reveal NGFRAP1's multifaceted functions, antibodies with enhanced specificity for different protein isoforms, post-translational modifications, and protein complexes will become increasingly valuable for advancing our understanding of this important signaling protein.