NANS Antibody

What is NANS and what is its biological function?

NANS (N-acetylneuraminate synthase) is an enzyme that produces N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN). It can also use N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively . The protein has ubiquitous tissue expression and plays a critical role in sialic acid biosynthesis pathways. Sialic acids are important components of glycoproteins and glycolipids involved in cellular recognition, signaling, and immune function. Understanding NANS expression and activity is valuable for research into glycobiology and related disorders.

What types of NANS antibodies are available for research applications?

Currently, the primary NANS antibodies available for research are rabbit polyclonal antibodies. These include antibodies targeting different epitope regions:

Both antibodies are raised against recombinant fragments of human NANS protein, making them particularly suitable for studies involving human samples . The different epitope targets allow researchers to select antibodies based on the specific region of interest or to use multiple antibodies for validation purposes.

What experimental applications are NANS antibodies suitable for?

NANS antibodies have been validated for several experimental applications:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Useful for localizing NANS in tissue sections, with successful staining demonstrated in human gastric cancer tissue. The protocol typically involves antigen retrieval using citrate buffer (pH 6.0) under high pressure followed by blocking with normal goat serum .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For detecting NANS in cultured cells, allowing subcellular localization studies .

• Western Blotting (WB): For detecting denatured NANS protein in cell or tissue lysates, with recommended dilutions of 1:500-1:2000 .

• ELISA: For quantitative measurement of NANS protein, starting with a recommended concentration of 1 μg/mL that can be optimized based on specific assay requirements .

What are the optimal protocols for using NANS antibodies in immunohistochemistry?

For optimal IHC-P results with NANS antibodies, the following methodology has been validated:

• Tissue preparation: Dewax and rehydrate paraffin-embedded tissue sections following standard protocols.

• Antigen retrieval: Perform high-pressure antigen retrieval using citrate buffer (pH 6.0). This step is critical for exposing epitopes that may be masked during fixation.

• Blocking: Block sections with 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding.

• Primary antibody incubation: Dilute NANS antibody (e.g., ab236783) to 1/200 in 1% BSA solution and incubate at 4°C overnight.

• Detection: Use appropriate secondary antibody and detection system compatible with your visualization method .

This protocol has successfully demonstrated NANS expression in human gastric cancer tissue and can be adapted for other tissue types with appropriate controls.

How should researchers address developability issues with antibodies?

When working with NANS antibodies or developing new ones, researchers should consider:

• Early-stage assessment: Evaluate physicochemical properties affecting homogeneity, stability, solubility, and specificity during the discovery phase .

• Identification of PTM hotspots: Use in silico analysis to identify post-translational modification sites that could affect antibody performance, particularly in the CDR regions .

• Engineering solutions: If developability issues are identified, consider protein engineering approaches. For example:

  • Removal of deamidation sites in CDR regions

  • Elimination of proteolytic cleavage sites

  • Optimization to reduce polyreactivity and self-association potential

• Formulation optimization: For antibodies with minor stability issues like visible particles or precipitates, test different formulation buffers before resorting to sequence engineering .

What controls should be included when using NANS antibodies?

To ensure reliable and interpretable results with NANS antibodies, implement the following controls:

• Positive control: Include tissues or cell lines known to express NANS (since NANS is ubiquitously expressed, human gastric tissue has been validated) .

• Negative control: Use one of the following:

  • Primary antibody omission

  • Isotype control antibody at the same concentration

  • Pre-absorption with the immunizing peptide if available

• Technical controls:

  • Include internal controls within the same sample when possible

  • Use standardized protocols with consistent antibody lots

  • Include loading controls for Western blot applications

• Validation controls: Consider orthogonal methods to confirm antibody specificity, such as using a second antibody targeting a different epitope of NANS.

How can researchers optimize antibody dilutions for different applications?

For optimal NANS antibody performance, a systematic titration approach is recommended:

• Starting dilutions: Begin with the manufacturer's recommended dilutions:

  • For IHC-P: 1/200 dilution has been validated for ab236783

  • For WB: 1:500-1:2000 range for STJ11100751

  • For ELISA: Start at 1 μg/mL for STJ11100751

• Titration strategy:

  • Prepare a series of 2-fold or 3-fold dilutions around the recommended concentration

  • Test on positive control samples

  • Evaluate signal-to-noise ratio and specificity at each dilution

• Optimization factors:

  • Sample type and preparation method

  • Detection system sensitivity

  • Incubation time and temperature

  • Buffer composition

• Validation: Once optimized, confirm reproducibility across multiple experiments.

What are common issues with NANS antibodies and how can they be resolved?

How can researchers use NANS antibodies to study post-translational modifications?

To investigate post-translational modifications (PTMs) of NANS:

• Combined antibody approach: Use NANS antibodies alongside PTM-specific antibodies (phospho-specific, glycosylation-specific, etc.) in sequential or dual immunostaining.

• Immunoprecipitation strategy:

  • Immunoprecipitate NANS using validated antibodies

  • Perform Western blot analysis with PTM-specific antibodies

  • Alternatively, use mass spectrometry to identify modifications on the immunoprecipitated protein

• Functional studies:

  • Compare wild-type NANS activity with mutated versions where potential PTM sites are altered

  • Use NANS antibodies to track protein localization and expression levels

• Temporal analysis:

  • Use NANS antibodies to monitor changes in expression or localization following stimuli that might induce PTMs

  • Correlate with enzyme activity measurements

What considerations should researchers make when studying NANS in disease models?

When investigating NANS in disease contexts:

• Expression analysis: Use validated NANS antibodies for comparative expression studies between normal and diseased tissues. The NANS antibody ab236783 has been successfully used to detect NANS in gastric cancer tissue .

• Specificity verification: Verify antibody specificity in your specific disease model, as altered protein conformations or interactions in disease states may affect epitope accessibility.

• Correlation with function: Combine antibody-based detection of NANS with functional assays measuring sialic acid production to establish relationships between expression and activity.

• Cross-species considerations: When working with animal models, confirm cross-reactivity of your NANS antibody with the species under study. Current antibodies have been primarily validated with human samples .

• Context-specific controls: Include both normal and disease-specific controls relevant to your research question.

How do researchers interpret conflicting results from different NANS antibodies?

When faced with discrepant results using different NANS antibodies:

• Epitope mapping: Consider the specific regions targeted by each antibody (e.g., aa 100-300 vs. aa 260-359) . Different epitopes may be differentially accessible depending on:

  • Protein conformation

  • Protein-protein interactions

  • Post-translational modifications

  • Fixation or denaturation methods

• Validation approach:

  • Use orthogonal techniques (mRNA expression, activity assays) to corroborate protein expression data

  • Test antibodies on samples with known NANS expression levels

  • Consider using genetic tools (CRISPR knockout, siRNA) to validate specificity

• Technical optimization:

  • Test both antibodies under identical conditions when possible

  • Optimize protocols separately for each antibody

  • Consider the detection methods and their sensitivity

• Integrated analysis: Rather than dismissing conflicting results, use them to develop a more complete understanding of NANS biology, as they may reveal context-dependent protein conformations or interactions.

How can NANS antibodies be utilized in high-throughput screening approaches?

NANS antibodies can be adapted for high-throughput applications through:

• Automated immunoassay platforms:

  • Develop ELISA-based high-throughput screening using validated NANS antibodies (e.g., STJ11100751)

  • Implement robotics for sample preparation, antibody addition, and washing steps

  • Use plate readers for rapid detection and analysis

• Tissue microarray analysis:

  • Apply NANS antibodies validated for IHC (e.g., ab236783) to tissue microarrays

  • Combine with digital pathology for automated scoring

  • Enable screening of NANS expression across multiple tissue types or patient samples simultaneously

• Flow cytometry applications:

  • Adapt NANS antibodies for intracellular staining in flow cytometry

  • Enable rapid screening of cell populations for NANS expression

  • Combine with other markers for multiparameter analysis

• Considerations for optimization:

  • Antibody specificity becomes even more critical in high-throughput contexts

  • Include appropriate controls on each plate/array

  • Validate consistency across batches

What is the role of NANS antibodies in studying the relationship between sialic acid metabolism and disease?

NANS antibodies provide valuable tools for investigating sialic acid metabolism in disease contexts:

• Cancer research: Monitor NANS expression in different cancer types to understand alterations in sialic acid metabolism, which may contribute to cancer cell immune evasion and metastasis. The successful application of NANS antibody in gastric cancer tissue demonstrates this potential .

• Neurodegenerative diseases: Investigate NANS expression in relation to altered sialylation patterns observed in conditions like Alzheimer's disease.

• Congenital disorders of glycosylation: Use NANS antibodies to study protein expression and localization in rare genetic disorders affecting the sialic acid pathway.

• Inflammatory conditions: Examine how changes in NANS expression correlate with alterations in sialylated glycans that modulate immune responses.

• Research methodology:

  • Compare NANS protein levels with enzyme activity measurements

  • Correlate with downstream sialylated glycan profiles

  • Use in combination with genetic analyses to understand the impact of NANS variants