NBR1 Monoclonal Antibody

What is NBR1 protein and what are its key structural features?

NBR1 is a ubiquitously expressed 988-amino acid multidomain scaffold protein that shows approximately 90% conservation between mouse and human species . Originally identified as an ovarian tumor antigen, NBR1 contains several important structural domains that contribute to its diverse functions:

• N-terminal phox/Bem1p (PB1) domain

• ZZ-type zinc finger (ZZ)

• Coiled-coiled (CC) region

• C-terminal ubiquitin association (UBA) domain capable of binding to both K48- and K63-type polyubiquitin chains

The protein contains a B-box/coiled coil motif, which is present in many genes with transformation potential . NBR1 is located on chromosome 17q21.1, in close proximity to the tumor suppressor gene BRCA1, and has three alternatively spliced variants encoding the same protein . The calculated molecular weight is 107 kDa, but the observed molecular weight in experimental conditions is approximately 140 kDa .

What applications are NBR1 monoclonal antibodies suitable for?

NBR1 monoclonal antibodies have been validated for multiple research applications with specific dilution recommendations for optimal results:

The antibody's versatility across multiple applications makes it valuable for comprehensive protein characterization . For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may serve as an alternative .

What are the recommended storage and handling conditions for NBR1 antibodies?

Proper storage and handling are critical for maintaining antibody performance over time:

• Storage temperature: Store at -20°C in aliquots to minimize freeze-thaw cycles .

• Buffer composition: NBR1 antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.4 .

• Stability: When stored properly, NBR1 antibodies remain stable for one year after shipment .

• Aliquoting: While some formulations indicate aliquoting is unnecessary for -20°C storage, dividing into small volumes is generally recommended to avoid repeated freeze-thaw cycles that can compromise antibody performance .

• Working dilutions: Prepare fresh working dilutions on the day of the experiment for optimal results.

Antibody formulations may contain small amounts of BSA (0.1%) in smaller volume preparations (20 μl), which helps maintain stability .

How should researchers validate the specificity of NBR1 antibodies?

Validating antibody specificity is essential for reliable research findings:

• Positive controls: Use cell lines with known NBR1 expression such as HeLa and COS-7 cells for western blot applications .

• Molecular weight verification: Confirm the observed band appears at the expected molecular weight of approximately 140 kDa .

• Knockdown/knockout validation: Multiple publications have used siRNA or CRISPR techniques to validate NBR1 antibody specificity through depletion of the target protein .

• Cross-reactivity assessment: Test the antibody against samples from different species if cross-species reactivity is claimed. The NBR1 antibody has demonstrated reactivity with human, mouse, rat, and monkey samples .

• Immunogen consideration: Understanding the immunogen used (e.g., NBR1 fusion protein) provides insight into potential cross-reactivity and epitope recognition .

How does NBR1 function in receptor tyrosine kinase (RTK) trafficking pathways?

NBR1 plays a critical regulatory role in RTK trafficking and degradation pathways:

• Inhibitory function: NBR1 inhibits ligand-mediated lysosomal degradation of RTKs, likely by inhibiting receptor internalization from the cell surface .

• Domain requirements: The C-terminus of NBR1 is essential but not sufficient for this inhibitory activity on RTK degradation .

• Experimental evidence: Research shows that ectopic NBR1 expression inhibits ligand-mediated lysosomal degradation of endogenous RTKs, while siRNA depletion of endogenous NBR1 enhances receptor degradation .

• Mechanism insights: Live-cell imaging reveals that NBR1's effect on RTK degradation is likely due to inhibition of receptor internalization from the cell surface rather than effects on later trafficking steps .

• Localization features: While the C-terminus contains a membrane-interacting amphipathic α-helix necessary for late endocytic localization, this feature is not required for NBR1's effect on RTK degradation, suggesting separate functional domains .

For researchers studying RTK trafficking, using NBR1 antibodies in combination with RTK-specific antibodies in co-localization experiments can provide valuable insights into the spatial and temporal dynamics of these interactions.

What is NBR1's role in selective autophagy and how can researchers study this function?

NBR1 serves as an autophagy receptor with distinct functional attributes:

• Autophagy adaptor function: NBR1 acts as a specific adaptor for ubiquitinated cargos destined for degradation by autophagosomes, similar to P62/SQSTM1 .

• Key interaction domains: NBR1 associates with LC3 (microtubule-associated protein 1 light chain 3), the mammalian homolog of Atg8, through a main LC3-interacting region (LIR) and a secondary LC3-interacting region (LIR2) .

• Experimental approaches:

  • Use NBR1 antibodies in co-immunoprecipitation experiments to identify interaction partners

  • Apply immunofluorescence with NBR1 antibodies to visualize co-localization with autophagosomal markers

  • Employ Western blot analysis to monitor NBR1 levels during autophagy induction/inhibition

• Important distinction: Research indicates that the late endocytic and autophagic localizations of NBR1 are independent of one another, suggesting that NBR1's functions in each context might be distinct .

For studying NBR1 in autophagy, researchers should consider using autophagy modulators (such as rapamycin or bafilomycin A1) in combination with NBR1 immunodetection to observe dynamic changes in NBR1 localization and interaction patterns.

How can researchers optimize NBR1 antibody use in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with NBR1 antibodies requires careful optimization:

• Lysate preparation: Use gentle lysis buffers (containing 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions. The recommended protein amount is 1.0-3.0 mg of total protein lysate with 0.5-4.0 μg of antibody .

• Pre-clearing step: Implement a pre-clearing step with appropriate control IgG to reduce non-specific binding.

• Known interactors to validate Co-IP:

  • P62/SQSTM1, which heterodimerizes with NBR1 via the PB1 domain

  • Spred2, a negative regulator of ERK1/2 signaling downstream of RTKs

  • LC3, which interacts with NBR1 during autophagy

• Controls: Include appropriate negative controls (non-immune IgG) and positive controls (input lysate) in each experiment.

• Detection strategies: For western blot detection after Co-IP, adjust antibody dilution to 1:500 to enhance sensitivity for potentially low-abundance co-precipitated proteins .

When studying novel NBR1 interactions, consider crosslinking approaches to stabilize transient interactions before cell lysis and immunoprecipitation.

What are the optimal conditions for immunohistochemical detection of NBR1 in tissue samples?

Successful immunohistochemical detection of NBR1 requires specific technical considerations:

• Antigen retrieval: The preferred method is heat-induced epitope retrieval using TE buffer at pH 9.0, although citrate buffer at pH 6.0 may be used as an alternative .

• Tissue fixation: Formalin-fixed, paraffin-embedded tissues are suitable for NBR1 immunodetection.

• Dilution optimization: Begin with a 1:100 dilution and adjust based on signal intensity and background levels. The recommended range is 1:50-1:500 .

• Positive control tissues: Use mouse heart tissue or human breast cancer tissue as positive controls to validate staining protocols .

• Detection systems: Both chromogenic (DAB) and fluorescent secondary detection systems are compatible with NBR1 antibodies.

• Counterstaining: When using chromogenic detection, light hematoxylin counterstaining provides good nuclear contrast without obscuring NBR1 signals.

For multiplexed immunofluorescence studies involving NBR1, careful antibody panel design is essential to avoid species cross-reactivity when using multiple primary antibodies.

How can researchers differentiate between NBR1's roles in endocytic trafficking versus selective autophagy?

Distinguishing between NBR1's dual functions requires sophisticated experimental approaches:

• Compartment-specific markers: Use co-staining with markers for late endosomes (e.g., Rab7) versus autophagosomal markers (e.g., LC3) to differentiate localization patterns.

• Domain-specific mutations: Research has shown that:

  • The C-terminus of NBR1 is essential for late endocytic localization but not sufficient on its own

  • The C-terminal membrane-interacting amphipathic α-helix is necessary for late endocytic localization but not for NBR1's effect on RTK degradation

• Conditional perturbations:

  • Inhibit autophagy (using 3-methyladenine or ATG gene knockdowns) and assess NBR1's endocytic functions

  • Block endocytosis (using dynamin inhibitors) and examine NBR1's role in autophagy

• Live-cell imaging: Use fluorescently tagged NBR1 constructs in combination with immunofluorescence against endogenous NBR1 to track protein dynamics in real-time.

• Key insight: Research has demonstrated that late endocytic and autophagic localizations of NBR1 are independent of one another, suggesting distinct functions in each context .

By systematically manipulating each pathway and observing NBR1 behavior with antibody-based detection methods, researchers can untangle these overlapping but distinct functions.

What are the optimal conditions for using NBR1 antibodies in quantitative ELISA assays?

For reliable quantitative detection of NBR1 by ELISA:

• Sensitivity parameters: When used as a capture antibody in sandwich ELISA format, the NBR1 monoclonal antibody has a detection limit of approximately 0.1 ng/ml for recombinant GST-tagged NBR1 .

• Sample preparation: For cell/tissue lysates, use a buffer containing a mild detergent (0.1% Triton X-100) supplemented with protease inhibitors to prevent protein degradation.

• Standard curve generation: Use purified recombinant NBR1 protein at concentrations ranging from 0.1-100 ng/ml to establish a reliable standard curve .

• Antibody pairing: For sandwich ELISA, careful selection of capture and detection antibody pairs that recognize distinct, non-overlapping epitopes is essential.

• Validation approach: Confirm ELISA results with complementary techniques such as western blotting to verify specificity of detection.

When developing custom ELISA protocols, researchers should optimize blocking conditions and antibody concentrations through systematic titration experiments to minimize background while maximizing specific signal.

How should researchers interpret apparent molecular weight variations when detecting NBR1?

Understanding molecular weight discrepancies is crucial for accurate data interpretation:

• Expected versus observed weight: While the calculated molecular weight of NBR1 is 107 kDa based on its 966 amino acid sequence, the observed molecular weight in experimental conditions is typically around 140 kDa .

• Potential causes for discrepancy:

  • Post-translational modifications, particularly phosphorylation and ubiquitination

  • The presence of the highly structured domains affecting protein migration

  • Splice variants (three alternatively spliced variants encoding the same protein have been identified)

• Validation approaches:

  • Use denaturing conditions of varying stringency to confirm band identity

  • Employ size-exclusion chromatography to verify native protein size

  • Perform knockout/knockdown validation to confirm band specificity

• Detection recommendations: When performing western blot analysis, include molecular weight markers that span 100-150 kDa range for accurate size determination.

Researchers should note that different antibodies targeting distinct epitopes of NBR1 may show slight variations in the apparent molecular weight due to epitope accessibility in the denatured protein.

What experimental designs are recommended for studying NBR1 in the context of cancer research?

Given NBR1's proximity to BRCA1 and its identification as an ovarian tumor antigen, specific experimental approaches are valuable for cancer research:

• Tissue microarray analysis: Use NBR1 antibodies with 1:100-1:200 dilution for immunohistochemical screening across multiple tumor types and matched normal tissues .

• Expression correlation studies:

  • Compare NBR1 and BRCA1 expression patterns in breast and ovarian cancer tissues

  • Correlate expression with patient outcome data and treatment response

• Functional studies:

  • Examine the impact of NBR1 knockdown on cancer cell proliferation, migration, and response to therapy

  • Investigate NBR1's role in receptor tyrosine kinase trafficking in cancer cells with aberrant RTK signaling

• Co-localization analyses: Perform immunofluorescence studies to examine NBR1 localization relative to:

  • Growth factor receptors commonly overexpressed in cancers (EGFR, HER2)

  • Autophagic markers in nutrient-deprived cancer cells

• Cell line models: HeLa cells have been validated for NBR1 antibody applications and serve as a useful model for cancer-related studies .

When designing cancer-focused experiments, consider combining NBR1 antibody detection with markers of key cancer-related processes such as proliferation, apoptosis, and metastasis to establish functional correlations.

How can researchers address weak or inconsistent signal issues in Western blot applications?

When encountering detection challenges with NBR1 antibodies in Western blotting:

• Sample preparation optimization:

  • Ensure complete protein denaturation by heating samples at 95°C for 5 minutes in SDS-containing buffer

  • Use fresh protease inhibitors in lysis buffers to prevent degradation

  • Consider using RIPA buffer for more efficient extraction of membrane-associated proteins

• Transfer efficiency improvement:

  • For high molecular weight proteins like NBR1 (140 kDa), extend transfer time or reduce voltage

  • Consider using specialized transfer systems designed for high molecular weight proteins

• Antibody concentration adjustment:

  • Increase primary antibody concentration to 1:500 if signal is weak at 1:1000 dilution

  • Extend primary antibody incubation to overnight at 4°C

• Detection enhancement:

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Consider signal amplification systems for very low abundance targets

• Positive control inclusion: Always run lysates from HeLa or COS-7 cells as positive controls, as these have been validated for NBR1 detection .

If problems persist, consider using alternative lysate preparation methods or testing for potential interference from buffer components.

What strategies can improve NBR1 detection in fixed tissue immunohistochemistry?

To enhance NBR1 detection in tissue sections:

• Optimized antigen retrieval:

  • Test both recommended methods: TE buffer at pH 9.0 (preferred) and citrate buffer at pH 6.0 (alternative)

  • Extend retrieval time for heavily fixed tissues (up to 30 minutes)

• Signal amplification options:

  • Employ polymer-based detection systems for enhanced sensitivity

  • Consider tyramide signal amplification for very low abundance targets

• Background reduction:

  • Include additional blocking steps with animal serum matching the secondary antibody host

  • Use avidin/biotin blocking for tissues with high endogenous biotin

  • Include specific blocking of endogenous peroxidase activity

• Concentration optimization:

  • Begin with 1:100 dilution and adjust based on signal-to-noise ratio

  • The recommended range of 1:50-1:500 provides flexibility for optimization

• Incubation modifications:

  • Extend primary antibody incubation to overnight at 4°C for enhanced sensitivity

  • Consider the use of humidity chambers to prevent section drying

For particularly challenging tissues, preliminary experiments comparing fresh frozen sections with FFPE material may help determine optimal fixation conditions for NBR1 detection.

What emerging techniques might enhance NBR1 protein interaction studies?

As research on NBR1 continues to evolve, several advanced methodologies offer promising approaches:

• Proximity labeling methods:

  • BioID or TurboID fusion with NBR1 to identify proximity interactions in living cells

  • APEX2-based proximity labeling to capture transient interactions in specific cellular compartments

• Advanced microscopy approaches:

  • Super-resolution microscopy (STORM, PALM) to visualize NBR1 within subcellular structures at nanoscale resolution

  • Live-cell FRET imaging to monitor dynamic NBR1 interactions with partners like LC3 or RTKs

• Cryo-electron microscopy:

  • Structural determination of NBR1 complexes to understand molecular mechanisms of interaction

  • Visualization of NBR1's association with membrane structures

• Proteomics integration:

  • Quantitative interaction proteomics comparing NBR1 interactomes under various cellular conditions

  • Posttranslational modification mapping to understand regulatory mechanisms

• CRISPR-based approaches:

  • Endogenous tagging of NBR1 to study physiological levels of interaction

  • Domain-specific mutagenesis to dissect functional interactions

These emerging techniques, combined with traditional antibody-based approaches, will provide deeper insights into NBR1's multifaceted roles in cellular homeostasis.