NPTX1 Antibody, Biotin conjugated

What is NPTX1 and what are its functional roles in biological systems?

Neuronal pentraxin 1 (NPTX1), also known as NP1, is a 47 kDa protein that plays significant roles in neurological function and potentially in cancer pathways. At the molecular level, NPTX1 may mediate the uptake of degraded synaptic material, which could be critical in synaptic remodeling . Recent research has revealed that NPTX1 functions as a downstream target of the AKT pathway and can suppress tumor growth and promote mitochondria-related apoptosis in hepatocellular carcinoma (HCC) .

The protein has been identified as significantly downregulated in HCC tissues compared to adjacent normal tissues, with expression correlating with tumor size and metastatic potential . This dual role in both neurological function and cancer pathways makes NPTX1 an important research target across multiple disciplines.

What validated applications are suitable for NPTX1 antibody, biotin conjugated?

The biotin-conjugated NPTX1 antibody (Biotin-20656) has been specifically validated for immunohistochemistry (IHC) applications with demonstrated reactivity in human, mouse, and rat samples . The recommended dilution range for IHC applications is 1:200-1:800 .

For researchers requiring additional applications, the unconjugated NPTX1 antibody (20656-1-AP) has been validated for multiple applications as detailed in the following table:

It is strongly recommended that researchers titrate the antibody in their specific experimental system to determine optimal working conditions .

What is the recommended protocol for immunohistochemistry using biotin-conjugated NPTX1 antibody?

For optimal immunohistochemistry results with biotin-conjugated NPTX1 antibody, the following protocol is recommended:

• Sample preparation: Fix tissues with 10% formalin and embed in paraffin. Cut sections at 3-μm thickness .

• Deparaffinization and hydration: Follow standard protocols for deparaffinization and rehydration of sections.

• Antigen retrieval: Perform antigen retrieval using TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 may be used .

• Endogenous peroxidase blocking: Soak sections in 3% H₂O₂ at room temperature for 1 hour .

• Protein blocking: Block nonspecific binding proteins using appropriate blocking buffer.

• Primary antibody incubation: Apply biotin-conjugated NPTX1 antibody at a dilution of 1:200-1:800 in antibody diluent. Incubate at 4°C in a moist chamber overnight .

• Detection: Since the antibody is already biotin-conjugated, proceed directly to streptavidin-peroxidase conjugate incubation for 30 minutes at room temperature .

• Color development: Use 3,5-diaminobenzidine (DAB) for color development.

• Counterstaining: Counterstain with hematoxylin.

• Mounting: Dehydrate, clear, and mount sections.

Note that working conditions may need to be optimized for each laboratory's specific experimental setup.

How does NPTX1 expression correlate with cancer progression, and what considerations should be made in experimental design?

NPTX1 expression has significant implications for cancer research, particularly in hepatocellular carcinoma (HCC). Studies have shown that NPTX1 is downregulated in 64.15% of HCC tissues compared to adjacent non-tumor tissues . This altered expression correlates with critical clinical parameters:

• Tumor size: Lower NPTX1 expression correlates with larger tumor size in HCC patients .

• Metastatic potential: Decreased NPTX1 expression is associated with increased metastatic capability .

For experimental design, researchers should consider:

• Cell line selection: Different HCC cell lines demonstrate varying levels of NPTX1 expression. SMMC-7721 and MHCC-97h show relatively low expression, making them suitable for overexpression studies, while normal liver cell line LO2 expresses higher levels of NPTX1 .

• Functional assays: When investigating NPTX1's role, incorporate proliferation assays (such as CCK-8), colony formation assays, and apoptosis assays to comprehensively assess its tumor-suppressive functions .

• Cell cycle analysis: Flow cytometry for cell cycle distribution is essential as NPTX1 has been shown to induce G₀/G₁ phase arrest in HCC cells .

• Molecular pathway analysis: Include Western blot analysis of cell cycle-related proteins (CDK2, CDK4, CDK6, Cyclin A2, Cyclin D2) and AKT pathway components to elucidate mechanism .

When designing experiments that manipulate NPTX1 expression, validated overexpression constructs and knockdown approaches have been established. For NPTX1 overexpression, a 1299-bp genomic sequence of the NPTX1 coding region inserted into the pLV-puro plasmid has been used successfully. For knockdown, shRNA targeting NPTX1 (target sequence: 5′-GATCCGCAAACTTTGCAATCGCTCAACTCGAGTTGAGCGATTGCAAAGTTTGCTTTTTG-3′) inserted into the pLV-shRNA-puro plasmid has shown efficacy .

What approaches can be used to enhance specificity and sensitivity when detecting NPTX1 in tissue samples?

Enhancing specificity and sensitivity for NPTX1 detection requires careful consideration of several technical factors:

• Antibody selection: Choose antibodies validated for specific applications. The biotin-conjugated NPTX1 antibody (Biotin-20656) is specifically validated for IHC applications, while unconjugated antibody (20656-1-AP) offers broader application potential .

• Antigen retrieval optimization: Compare results using both recommended methods:

  • TE buffer at pH 9.0 (primary recommendation)

  • Citrate buffer at pH 6.0 (alternative method)

• Blocking optimization: Thorough blocking of nonspecific binding sites is crucial. For biotin-conjugated antibodies, additional avidin/biotin blocking may be necessary to reduce background caused by endogenous biotin.

• Signal amplification considerations: While biotin-conjugated antibodies provide natural signal amplification through avidin-biotin interaction, there may be trade-offs with background levels. Titration experiments (testing multiple dilutions) should be performed to determine optimal signal-to-noise ratio.

• ELISA-based detection: For quantitative measurement of NPTX1 in solution samples, sandwich ELISA offers high sensitivity with detection ranges of 7.8-500 pg/ml and a lower limit of detection of approximately 1.95 pg/ml . The assay employs:

  • Pre-coated microplates with NPTX1-specific antibody

  • Biotin-conjugated detection antibody

  • Avidin-HRP conjugate

  • Substrate solution for colorimetric detection

• Validation strategies: To confirm specificity, include appropriate controls:

  • Positive control tissues (mouse brain tissue for IHC)

  • Negative controls (omission of primary antibody)

  • Competitive inhibition with immunizing peptide

How can researchers effectively investigate NPTX1's role in the AKT signaling pathway in cancer models?

NPTX1 has been identified as a downstream target of the AKT pathway in hepatocellular carcinoma, with significant implications for tumor growth and apoptosis regulation . To effectively investigate this relationship, researchers should consider the following methodological approach:

• Expression correlation analysis:

  • Assess the correlation between NPTX1 expression and AKT pathway activation markers in patient samples and cell lines

  • Analyze phosphorylated AKT (p-AKT) levels in relation to NPTX1 expression using Western blot and immunohistochemistry

• Pathway manipulation experiments:

  • AKT inhibition: Treat cells with AKT pathway inhibitors (e.g., LY294002) to determine effects on NPTX1 expression

  • AKT activation: Use constitutively active AKT constructs to examine impact on NPTX1 levels

  • Combined manipulation: Simultaneously manipulate NPTX1 (overexpression/knockdown) and AKT pathway status to assess functional interaction

• Functional readouts:

  • Cell proliferation assays (CCK-8 assay or similar)

  • Apoptosis assays (flow cytometry with Annexin V/PI staining)

  • Cell cycle analysis using flow cytometry (particularly focusing on G₀/G₁ arrest)

  • Colony formation assays

• Molecular mechanism investigation:

  • Track changes in cell cycle regulatory proteins (CDK2, CDK4, CDK6, Cyclin A2, Cyclin D2)

  • Assess mitochondrial apoptosis markers

  • Evaluate changes in downstream AKT targets together with NPTX1 manipulation

Previous research has demonstrated that NPTX1's tumor-suppressive effects are significantly enhanced after blocking the AKT pathway, suggesting a regulatory relationship between AKT signaling and NPTX1 function . This provides a strong foundation for hypotheses regarding NPTX1's position in the signaling cascade.

What technical considerations should be observed when using biotin-conjugated NPTX1 antibody in multiplex immunohistochemistry?

Multiplex immunohistochemistry with biotin-conjugated NPTX1 antibody presents several technical challenges that require careful methodological considerations:

• Endogenous biotin blocking: Tissue samples, particularly liver tissues, contain significant levels of endogenous biotin that can cause high background. Use commercial avidin/biotin blocking kits before applying biotin-conjugated antibodies.

• Detection system selection: When using biotin-conjugated NPTX1 antibody with other antibodies in multiplex staining:

  • Avoid using multiple biotin-conjugated antibodies

  • Consider fluorescence-based multiplex systems where biotin-conjugated NPTX1 can be detected with streptavidin-conjugated fluorophores

  • For chromogenic multiplexing, position the biotin-conjugated antibody detection as either the first or last step in the sequence

• Cross-reactivity prevention:

  • Carefully select primary antibodies raised in different host species

  • When using multiple rabbit antibodies including biotin-conjugated NPTX1 (rabbit origin), implement sequential staining with complete antibody stripping or blocking between rounds

• Optimization of antibody dilution: The recommended dilution range for biotin-conjugated NPTX1 antibody is 1:200-1:800 for IHC applications . For multiplex applications, typically use the higher end of the dilution range to minimize background.

• Antigen retrieval considerations:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Ensure compatibility of retrieval conditions with other targets in multiplex panel

• Storage and stability considerations: Store the biotin-conjugated NPTX1 antibody at -20°C and avoid repeated freeze-thaw cycles. The antibody is stable for one year after shipment when properly stored . Protect from light exposure to maintain the integrity of the biotin conjugate.

• Controls for multiplex validation:

  • Single stain controls for each antibody to establish baseline staining pattern

  • Staining controls with omission of individual primary antibodies

  • Spectral unmixing controls when using fluorescence detection systems

What are common challenges in NPTX1 detection and how can they be addressed?

Researchers working with NPTX1 antibodies may encounter several technical challenges. Here are common issues and recommended solutions:

• High background in IHC applications:

  • Cause: Insufficient blocking, excessive antibody concentration, or endogenous biotin

  • Solution: Increase blocking time, optimize antibody dilution (try 1:400-1:800 range for biotin-conjugated antibody), and implement avidin-biotin blocking steps

• Weak or absent signal:

  • Cause: Suboptimal antigen retrieval, inappropriate antibody dilution, or low target expression

  • Solution: Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) antigen retrieval, reduce antibody dilution within recommended range, extend primary antibody incubation to overnight at 4°C

• Inconsistent results across experiments:

  • Cause: Variations in tissue processing, storage conditions, or antibody handling

  • Solution: Standardize fixation protocols (10% formalin recommended), avoid repeated freeze-thaw cycles of antibody, aliquot antibody for single use, and store at -20°C

• Cross-reactivity concerns:

  • Cause: Antibody binding to related proteins

  • Solution: Include appropriate negative controls, validate results with alternative detection methods (e.g., comparison of IHC with Western blot), and consider knockdown/knockout controls to confirm specificity

• Quantification challenges:

  • Cause: Subjective interpretation of staining intensity

  • Solution: Implement digital image analysis with standardized scoring systems, use quantitative methods like ELISA (with sensitivity of ~1.95 pg/ml) for soluble samples

How can researchers optimize experimental conditions for detecting NPTX1 in different tissue types?

Optimizing NPTX1 detection across different tissue types requires tailored approaches to account for tissue-specific characteristics:

• Brain tissue (high endogenous expression):

  • Use higher antibody dilutions (1:500-1:800 for biotin-conjugated antibody)

  • Apply shorter primary antibody incubation times (4-6 hours may be sufficient)

  • Mouse brain tissue serves as an excellent positive control

• Liver tissue (moderate expression, high background potential):

  • Implement extended blocking steps to reduce nonspecific binding

  • Careful avidin-biotin blocking is essential due to high endogenous biotin

  • HepG2 cells can serve as a positive control for Western blot applications

• Tumor tissues (variable expression):

  • NPTX1 is downregulated in hepatocellular carcinoma but expression may vary in other tumor types

  • Adjacent normal tissue should be included as internal control

  • Human gliomas tissue has been validated for IHC applications

• Special considerations for formalin-fixed paraffin-embedded (FFPE) tissues:

  • Extended antigen retrieval time may be necessary for older FFPE samples

  • Optimize section thickness (3-μm recommended)

  • Deparaffinization and hydration must be complete to ensure antibody access

• Fresh/frozen tissue considerations:

  • Adjust fixation protocols (shorter fixation times compared to FFPE)

  • Modify blocking and antibody concentrations (typically lower concentrations are effective)

• Tissue-specific dilution recommendations:

Remember that all recommended dilutions should be validated in each laboratory's specific experimental system .

How can NPTX1 antibodies be utilized in investigating neurodegenerative disease mechanisms?

While the search results focus primarily on NPTX1's role in cancer research, its native function in neuronal tissues suggests significant potential for neurodegenerative disease research. The biotin-conjugated NPTX1 antibody can be particularly valuable in this context:

• Synaptic remodeling studies: NPTX1 mediates uptake of degraded synaptic material, playing a potentially crucial role in synaptic remodeling . Researchers can use the antibody to:

  • Visualize NPTX1 distribution at synapses using high-resolution microscopy

  • Track changes in NPTX1 localization during disease progression

  • Correlate NPTX1 expression with synaptic markers in models of neurodegeneration

• Brain region-specific expression analysis: The biotin-conjugated antibody's validation for IHC in brain tissue enables:

  • Mapping of regional NPTX1 expression across healthy and diseased brain tissues

  • Correlation of expression patterns with pathological hallmarks of neurodegenerative diseases

  • Identification of vulnerable neuronal populations based on NPTX1 expression

• Co-localization with disease markers: In multiplex IHC applications, researchers can investigate:

  • NPTX1 co-localization with amyloid plaques or tau tangles in Alzheimer's disease models

  • Relationship between NPTX1 and α-synuclein aggregates in Parkinson's disease

  • Potential role in neuroinflammatory processes through co-staining with glial markers

• Functional manipulation studies: Combining antibody-based detection with genetic manipulation approaches:

  • Track changes in NPTX1 localization after pharmacological interventions

  • Correlate protein expression with behavioral outcomes in animal models

  • Investigate potential compensatory mechanisms involving related pentraxin family proteins

• Technical optimization for neuronal tissues:

  • For fresh brain slices, reduce antibody concentrations to 1:500-1:800

  • For fixed brain tissues, extended antibody incubation (overnight at 4°C) is recommended

  • Fluorescent detection systems may provide better signal-to-noise ratio in brain tissue than chromogenic methods

What emerging technologies can enhance the utility of NPTX1 antibodies in cancer research?

Several emerging technologies can significantly extend the research applications of NPTX1 antibodies, particularly the biotin-conjugated variant, in cancer research:

• Single-cell analysis techniques:

  • Combine biotin-conjugated NPTX1 antibody with mass cytometry (CyTOF) for high-dimensional single-cell protein expression analysis

  • Integrate with single-cell RNA sequencing data to correlate protein and transcript levels at single-cell resolution

  • Apply in spatial transcriptomics platforms to map NPTX1 protein expression in the context of the tumor microenvironment

• Advanced imaging approaches:

  • Implement super-resolution microscopy to visualize subcellular localization of NPTX1

  • Apply multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) for highly multiplexed tissue imaging

  • Utilize whole-slide digital pathology with AI-assisted quantification for large-scale analysis of NPTX1 expression across tumor cohorts

• Liquid biopsy applications:

  • Develop protocols for detecting NPTX1 in circulating tumor cells using the biotin-conjugated antibody

  • Explore potential for NPTX1 as a biomarker in extracellular vesicles (exosomes) derived from tumor cells

  • Integrate with other cancer biomarkers in multiplex detection systems

• Therapeutic targeting strategies:

  • Use antibodies to validate NPTX1 as a potential therapeutic target in cancers where it functions as a tumor suppressor

  • Investigate the AKT-NPTX1 axis as a potential combinatorial therapeutic approach

  • Develop tools to monitor NPTX1 expression as a pharmacodynamic biomarker for AKT pathway inhibitors

• Organoid and 3D culture applications:

  • Optimize immunofluorescence protocols using biotin-conjugated NPTX1 antibody for 3D culture systems

  • Track NPTX1 expression changes during organoid development and in response to drugs

  • Correlate expression patterns with invasive properties in 3D models

These emerging technologies, combined with the specificity of biotin-conjugated NPTX1 antibody, can significantly expand our understanding of NPTX1's role in cancer biology and potentially identify new therapeutic strategies targeting the AKT-NPTX1 axis .