NRSN1 Antibody

What is NRSN1 and why are antibodies against it important for research?

NRSN1 (Neurensin-1), also known as Neuro-p24 or vesicular membrane protein p24, is a neural-specific protein with a molecular weight of approximately 21.5 kDa. It plays important roles in neural organelle transport, transduction of nerve signals, neurite extension, and may contribute to memory consolidation .

NRSN1 antibodies are critical research tools for:

• Studying vesicular transport mechanisms in neurons

• Investigating neurite extension and neural development

• Examining potential roles in memory and cognition

• Exploring NRSN1 as a potential therapeutic target in certain cancers

What applications are NRSN1 antibodies validated for in research settings?

NRSN1 antibodies have been validated for several key applications in research:

The optimal dilution should be determined empirically for each application and specific antibody .

How should NRSN1 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of NRSN1 antibodies are crucial for maintaining their specificity and sensitivity:

• Storage temperature: Store at -20°C for long-term storage or 4°C for up to one month for frequent use

• Formulation: Most commercial NRSN1 antibodies are supplied in liquid form containing PBS with glycerol (typically 40-50%) and a preservative (such as 0.02% sodium azide)

• Aliquoting: Divide into small aliquots upon receipt to avoid repeated freeze-thaw cycles, which can decrease antibody activity

• Working solutions: Dilute in appropriate buffer immediately before use

• Shelf life: Most manufacturers guarantee activity for at least 12 months when stored properly

What controls should be used when working with NRSN1 antibodies?

Appropriate controls are essential for validating NRSN1 antibody specificity:

• Positive control: Tissues or cell lines known to express NRSN1 (neural tissues, SHP77 and NCI-H526 SCLC cell lines)

• Negative control: Non-neural tissues or HEK293 cells (show little NRSN1 expression)

• Blocking peptide: Using recombinant NRSN1 protein fragments (such as aa 140-195) for competitive blocking experiments

• NRSN1 knockout: Testing the antibody on NRSN1 knockout samples provides the most stringent control for specificity

• Isotype control: Using non-specific IgG from the same host species and at the same concentration

How can NRSN1 antibodies be used to study vesicular transport in neurons?

Investigating vesicular transport using NRSN1 antibodies requires specialized approaches:

• Co-localization studies:

  • Perform double immunofluorescence with NRSN1 antibodies and markers of vesicular compartments (synaptophysin, VAMP2)

  • Use super-resolution microscopy (STORM, STED) for precise spatial localization

  • Quantify co-localization using Pearson's or Mander's coefficients

• Live imaging of vesicular transport:

  • Generate NRSN1-GFP fusion constructs for live imaging

  • Use antibody-based proximity labeling methods (APEX, BioID) to identify NRSN1 interaction partners

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to study dynamics

• Biochemical fractionation:

  • Use NRSN1 antibodies in immunoblotting of synaptic vesicle fractions

  • Perform immunoprecipitation followed by mass spectrometry to identify vesicular cargo proteins

  • Conduct gradient ultracentrifugation with subsequent immunodetection of NRSN1

What are the emerging applications of NRSN1 antibodies in cancer research?

Recent research has identified NRSN1 as a potential target for antibody-drug conjugates (ADCs) in small cell lung cancer (SCLC) treatment:

• Expression profiling:

  • NRSN1 was found to be overexpressed specifically in SCLC with little to no expression in normal tissues

  • Cell surface expression was confirmed using flow cytometry in SCLC cell lines (SHP77 and NCI-H526)

  • Expression levels correlate with mRNA levels, with SHP77 showing highest expression, NCI-H526 moderate expression, and HEK293 minimal expression

• Therapeutic potential:

  • The combination of primary anti-NRSN1 monoclonal antibody and a secondary ADC exhibited anti-tumor activity in SCLC cell lines

  • CRISPR/Cas9-mediated knockout of NRSN1 in SHP77 cells resulted in loss of the anti-tumor activity, confirming specificity

  • Future development may involve direct conjugation of cytotoxic agents to anti-NRSN1 antibodies, optimizing drug-antibody ratios, and exploring different linker chemistries

• Methodological approaches:

  • Flow cytometry to quantify cell surface expression of NRSN1

  • Cell viability assays to assess cytotoxic effects of NRSN1-targeted ADCs

  • qRT-PCR for NRSN1 expression analysis using specific primers (forward: 5′-GAT TCT TAC CAC AAC GGG CTA CA-3′, reverse: 5′-GGG TTT CAA AGG TGA TTG GGT C-3′)

How can researchers validate the specificity of NRSN1 antibodies?

Validation of NRSN1 antibody specificity requires multiple complementary approaches:

• Genetic validation:

  • CRISPR/Cas9-mediated knockout of NRSN1 should eliminate antibody staining

  • siRNA-mediated knockdown should reduce signal proportionally to knockdown efficiency

  • Overexpression systems can confirm increased signal with increased expression

• Biochemical validation:

  • Pre-incubation with recombinant NRSN1 protein (blocking peptide) should abolish staining

  • Western blot should show a band of appropriate molecular weight (~24 kDa)

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Cross-platform validation:

  • Correlation between protein detection (antibody-based) and mRNA expression (qRT-PCR)

  • Comparison of results using different antibody clones targeting distinct epitopes

  • Testing across multiple cell types with known expression patterns

What methodological considerations are important when using NRSN1 antibodies for multiplexed immunofluorescence?

Multiplexed detection involving NRSN1 antibodies requires special attention:

• Antibody compatibility:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • When using multiple rabbit antibodies, consider sequential staining with tyramide signal amplification

  • Validate each antibody individually before combining them

• Epitope retrieval optimization:

  • Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer composition (citrate vs. EDTA-based) and pH (6.0 vs. 9.0)

  • Determine optimal incubation times for retrieving NRSN1 without affecting other epitopes

• Signal separation strategies:

  • Use spectral unmixing for fluorophores with overlapping emission spectra

  • Consider linear unmixing algorithms for quantitative analysis

  • Employ appropriate controls to calculate and correct for autofluorescence

How do post-translational modifications of NRSN1 affect antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of NRSN1:

• Common PTMs affecting NRSN1:

  • Phosphorylation sites may alter protein conformation and epitope accessibility

  • Glycosylation may mask certain epitopes

  • Ubiquitination can affect protein stability and antibody recognition

• Methodological approaches:

  • Use phospho-specific antibodies to study activation states

  • Employ enzymatic treatments (phosphatases, glycosidases) prior to immunodetection

  • Combine with mass spectrometry to map modifications

• Considerations for experimental design:

  • Phosphatase inhibitors should be included in lysis buffers if studying phosphorylated forms

  • Different fixation methods may preserve PTMs differently

  • Cell stimulation conditions may alter PTM patterns

What are the best practices for quantitative analysis when using NRSN1 antibodies?

Accurate quantification requires rigorous methodological considerations:

• Western blot quantification:

  • Use validated loading controls appropriate for your experimental system

  • Establish a linear dynamic range for detection

  • Include standard curves using recombinant NRSN1 protein

  • Employ appropriate normalization strategies

• Immunofluorescence quantification:

  • Standardize image acquisition parameters (exposure, gain)

  • Use appropriate thresholding methods for signal segmentation

  • Perform background subtraction

  • Consider Z-stack acquisition for volumetric analysis

• Flow cytometry quantification:

  • Use fluorescence calibration beads to standardize measurements

  • Include appropriate isotype controls

  • Optimize staining concentrations to avoid saturation

  • Consider using median fluorescence intensity rather than mean for analysis