Nucb2 Antibody

What is NUCB2 and why is it important in research?

NUCB2 is an EF-hand Ca²⁺ binding protein that plays roles in multiple physiological processes including calcium homeostasis, hypothalamic regulation of feeding, and TNF receptor shedding. It's a 50 kDa protein that can be post-translationally processed into three fragments: nesfatin-1 (aa 1-82), nesfatin-2 (aa 85-163), and nesfatin-3 (aa 166-396) . The N-terminal nesfatin-1 fragment appears to contain most of the biological activity. NUCB2 has gained significant research interest due to its involvement in:

• Anorexigenic (appetite-suppressing) activity

• Gastric secretion regulation

• Role in glucose metabolism

• Potential involvement in cancer progression

NUCB2 is expressed in various tissues including the hypothalamus (particularly in the supraoptic nucleus and paraventricular nucleus), stomach, intestine, and other organs, making it relevant for diverse research areas from neuroscience to gastroenterology and oncology .

What types of NUCB2 antibodies are available for research applications?

Several types of NUCB2 antibodies are available for research, varying in host species, clonality, and target epitopes:

When selecting an antibody, researchers should consider the specific experimental requirements, including the species being studied, intended application, and target region of interest .

How does NUCB2 protein structure relate to antibody epitope selection?

NUCB2 contains several functional domains that influence antibody epitope selection:

• Signal peptide (24 aa) at N-terminus

• Nesfatin-1 domain (aa 25-106)

• DNA-binding domain (aa 171-223)

• EF-hand domain (aa 247-322)

The 51-75 aa region within the nesfatin-1 domain appears particularly important for biological function, as it contains a sequence that interacts with the melanocortin-4 receptor (MC4R) . When selecting antibodies, researchers should consider which domain is most relevant to their research question:

• For studies on appetite regulation, antibodies targeting the nesfatin-1 domain (particularly aa 51-75) may be most informative

• For investigating calcium binding, antibodies against the EF-hand domain may be preferable

• For general detection of NUCB2 expression, antibodies against conserved regions between species might be optimal

What are the optimal dilutions for NUCB2 antibodies in different applications?

Based on validated protocols, the recommended dilutions for NUCB2 antibodies vary by application:

It's important to note that these are starting recommendations, and antibody concentration should be titrated for each experimental system to obtain optimal results . Sample-dependent optimization is particularly important when working with different tissue types.

How should samples be prepared for optimal NUCB2 detection?

Sample preparation varies by technique and tissue type:

For Western Blotting:

• Protein extraction should be performed with buffers containing protease inhibitors to prevent degradation

• Phosphatase inhibitors should be included if investigating phosphorylated NUCB2

• The expected molecular weight range is 46-50 kDa for full-length NUCB2, with potential post-translationally modified forms at ~55 kDa in certain cancer cells

For Immunohistochemistry:

• Paraffin-embedded sections require antigen retrieval

• Primary recommendation: TE buffer pH 9.0

• Alternative: citrate buffer pH 6.0

• In gastric tissues, NUCB2 has been detected in secretory granules of chief cells and in the cytoplasm of parietal cells in functioning gastric glands

For cell culture samples:

• Positive ICC/IF detection has been validated in U2OS cells

• For gastric cancer research, SGC-7901 and AGS cell lines have shown detectable NUCB2 expression

How can I validate the specificity of my NUCB2 antibody?

Multiple validation approaches should be employed:

• Positive and negative controls:

  • Known positive tissues: mouse brain tissue, rat brain tissue, human milk

  • Cell lines: SGC-7901, U2OS cells

  • Negative controls should include secondary antibody-only controls and tissues known to lack NUCB2 expression

• Size confirmation:

  • Expected molecular weight range: 46-50 kDa for full-length NUCB2

  • In cancer samples, look for potential post-translationally modified forms (~55 kDa)

• RNAi knockdown:

  • Some antibodies (e.g., HPA008395) have been validated through RNAi knockdown experiments

  • This approach confirms that signal reduction follows NUCB2 expression reduction

• Multiple antibodies:

  • Use antibodies targeting different epitopes to confirm detection patterns

  • Compare monoclonal and polyclonal antibody results to ensure consistency

How can I distinguish between full-length NUCB2 and processed nesfatin-1 in my experiments?

Distinguishing between full-length NUCB2 and its processed fragments requires careful experimental design:

• Antibody selection:

  • Use antibodies specific to different regions of NUCB2

  • For nesfatin-1 specific detection, antibodies targeting aa 25-106 region

  • For full-length NUCB2, antibodies against C-terminal regions

• Western blot analysis:

  • Full-length NUCB2: ~50 kDa

  • Processed nesfatin-1: ~9.7 kDa

  • Use appropriate gel percentage (12-15% for nesfatin-1 detection)

• Important considerations:

  • Most commercial antibodies detect full-length NUCB2 but not mature nesfatin-1

  • The Phoenix Pharmaceuticals antibody has not detected endogenous processed nesfatin-1 in some studies

  • The purified nesfatin-1 ab24 antibody has successfully detected processed nesfatin-1 in rat cerebrospinal fluid

It's worth noting that there's some controversy regarding whether NUCB2 is cleaved to nesfatin-1 in vivo, as the unprocessed full-length NUCB2 appears biologically active. Many researchers now refer to the protein as "NUCB2/nesfatin-1" to acknowledge this uncertainty .

What methodological approaches can resolve contradictory findings in NUCB2 expression in cancer tissues?

Contradictory findings regarding NUCB2 expression in cancer require careful methodological consideration:

How can I investigate NUCB2 phosphorylation status in my experimental system?

NUCB2 phosphorylation has been documented in gastric mucosa and may be functionally significant:

• Detection methods:

  • Phospho-specific antibodies (if available)

  • Phospho-protein enrichment followed by Western blot with NUCB2 antibodies

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

• Experimental approach:

  • Compare phosphorylation status between normal and pathological tissues

  • Investigate kinases potentially involved using kinase inhibitors

  • Employ mass spectrometry to identify specific phosphorylation sites

• Functional assessment:

  • Correlate phosphorylation status with biological activity

  • Generate phosphomimetic and phospho-deficient mutants for functional studies

  • Study how phosphorylation affects protein-protein interactions, particularly with signaling partners

What is the significance of NUCB2 in gastric cancer progression?

Recent research has revealed complex roles for NUCB2 in gastric cancer:

• Clinical correlations:

  • High NUCB2 expression correlates with deep tumor invasion, lymphovascular invasion, lymph node metastasis, and advanced clinical stages

  • NUCB2 serves as an independent predictor of unfavorable progression-free survival in gastric cancer patients

  • Association with prognosis is particularly significant in patients who received post-operative chemotherapy

• Cellular mechanisms:

  • NUCB2 knockout induces cellular senescence in gastric cancer cell lines

  • This leads to increased E-cadherin expression and decreased proliferation

  • NUCB2 appears to inhibit senescence in gastric carcinoma, promoting tumor progression

  • Knockout experiments showed inhibited proliferation, increased susceptibility to apoptosis, and reduced migration capability

• Immune relevance:

  • NUCB2 has been identified as a potential tumor antigen eliciting autoantibody responses in 5.4% of gastric cancer patients

  • Autoantibodies against NUCB2 were found exclusively in gastric cancer patients, not in colon/breast cancer patients or healthy donors

  • The immunogenicity may be related to cancer-specific post-translational modifications or alternative transcript variants

How does NUCB2/nesfatin-1 function in metabolic regulation?

NUCB2/nesfatin-1 plays important roles in metabolic regulation through several mechanisms:

• Central nervous system actions:

  • Expression in hypothalamic nuclei involved in feeding regulation

  • Anorexigenic effects when injected into the third brain ventricle

  • The 51-75 amino acid region is crucial for binding to melanocortin-4 receptor (MC4R)

• Intestinal-hepatic axis:

  • Intestinal NUCB2/nesfatin-1 regulates hepatic glucose production

  • Functions via a gut-brain-liver circuit

  • May have implications for insulin sensitivity and glucose homeostasis

• Translational relevance:

  • Potential therapeutic target for metabolic disorders

  • Biomarker for investigating pathways in obesity and diabetes

  • Understanding of NUCB2/nesfatin-1 mechanisms could inform drug development strategies

What are the key methodological challenges in researching NUCB2 post-translational modifications?

Investigating NUCB2 post-translational modifications presents several methodological challenges:

• Detecting processed forms:

  • Controversy exists regarding whether NUCB2 is cleaved to nesfatin-1 in vivo

  • Different antibodies show different detection patterns

  • Need for specialized techniques to differentiate between full-length and processed forms

• Cancer-specific modifications:

  • A 55 kDa isoform has been detected in gastric tumors and AGS gastric cancer cells

  • This modification is absent in normal gastric mucosa

  • The nature of this modification remains unidentified but may trigger immune responses

  • Techniques like mass spectrometry are needed to characterize these modifications

• Phosphorylation analysis:

  • NUCB2 is phosphorylated in gastric mucosa

  • Phosphorylation may affect function, localization, or interaction with binding partners

  • Technical challenges include preserving phosphorylation during sample preparation

  • Need for phospho-specific antibodies or enrichment techniques

What novel applications of NUCB2 antibodies should researchers consider?

Several promising research directions emerge from recent findings:

• Cancer biomarker development:

  • Investigate NUCB2 as a prognostic marker in gastric cancer

  • Explore its role in other cancer types

  • Develop assays to detect cancer-specific post-translational modifications

• Therapeutic target validation:

  • Use antibodies to block specific domains to understand functional consequences

  • Investigate the 51-75 aa region as a potential therapeutic target for metabolic disorders

  • Develop approaches to modulate NUCB2-mediated cellular senescence in cancer

• Structural biology applications:

  • Use domain-specific antibodies to probe conformational changes

  • Investigate how NUCB2 interacts with binding partners

  • Study the structural basis for NUCB2's multiple biological functions

How can researchers resolve contradictions in the NUCB2 literature?

Addressing contradictions requires systematic methodological approaches:

• Standardized reporting:

  • Clear documentation of antibody specificity, including epitope information

  • Detailed sample preparation protocols

  • Comprehensive characterization of experimental models

• Multi-level analysis:

  • Integrate transcriptomics, proteomics, and functional data

  • Examine transcript variants and post-translational modifications

  • Consider tissue-specific and context-dependent effects

• Collaborative approaches:

  • Round-robin testing of antibodies across laboratories

  • Development of reference standards

  • Open data sharing to facilitate meta-analyses