UNC13B Antibody

What is UNC13B and why is it important in research?

UNC13B, also known as MUNC13-2 or Unc13h2, is a protein encoded by the UNC13B gene. This protein functions in carbohydrate metabolism, homeostasis, and chemical synaptic transmission. The human version of UNC13B has a canonical amino acid length of 1591 residues and a protein mass of 180.7 kilodaltons, with two identified isoforms . UNC13B is primarily localized in the cell membrane and cytoplasm, and is widely expressed across many tissue types . Research importance stems from its role in vesicle maturation and exocytosis, making it relevant to neuroscience, cell biology, and cancer research .

What are the key characteristics of UNC13B antibodies available for research?

UNC13B antibodies are available in various formats with different host species, clonality types, and applications. Most commercially available antibodies are polyclonal, developed in rabbit, mouse, or goat hosts . They typically target different epitopes, including N-terminal regions (AA 12-215, AA 1-350), internal regions, and C-terminal domains (AA 1482-1591) . These antibodies undergo antigen affinity purification to ensure specificity and are available in both conjugated and unconjugated forms depending on intended applications .

What are the validated applications for UNC13B antibodies?

UNC13B antibodies have been validated for multiple laboratory techniques as outlined in the table below:

Different antibodies may show varying performance across these applications, so validation for your specific experimental system is essential .

How should I optimize immunohistochemistry protocols for UNC13B detection?

When optimizing IHC for UNC13B detection, consider the following methodology:

• Antigen retrieval: Test both heat-induced epitope retrieval (citrate buffer pH 6.0) and enzymatic retrieval methods to determine optimal conditions.

• Antibody dilution: Begin with manufacturer-recommended dilutions (typically 1:30-1:150 for IHC) and perform a dilution series .

• Incubation conditions: Compare overnight incubation at 4°C versus 1-2 hours at room temperature.

• Detection system: Use high-sensitivity detection systems compatible with your primary antibody species.

• Controls: Include positive control tissues with known UNC13B expression and negative controls (omitting primary antibody).

• Counterstaining: Adjust hematoxylin timing to maintain visibility of UNC13B signal.

It's crucial to include both positive and negative controls to ensure specificity and avoid false positives caused by non-specific binding .

How can I investigate UNC13B's role in cancer drug resistance mechanisms?

To study UNC13B's involvement in drug resistance, particularly in contexts like arsenic trioxide (ATO) resistance in chronic myeloid leukemia, consider this methodological approach:

• Expression analysis: Compare UNC13B levels between drug-sensitive and resistant cell lines using validated antibodies in western blot and IHC applications .

• Functional studies: Use gene knockdown approaches (shRNA, siRNA) targeting UNC13B to assess changes in drug sensitivity .

• Proliferation and apoptosis assays: After UNC13B manipulation, conduct CCK-8 assays for proliferation and flow cytometry for apoptosis quantification .

• Colony formation assessment: Employ soft agar assays to evaluate tumor formation capacity with and without UNC13B knockdown .

• Pathway analysis: Investigate downstream effectors, particularly MAP3K7, CDK4, and PINK1, which have been identified as potential mediators of UNC13B effects on drug resistance .

Research has demonstrated that UNC13B downregulation significantly inhibits growth, promotes apoptosis, and decreases colony formation in ATO-resistant K-562 cells, suggesting that UNC13B may be a potential therapeutic target for ATO-resistant chronic myeloid leukemia .

What techniques should I use to distinguish between UNC13B isoforms?

Differentiating between the two known isoforms of UNC13B requires careful experimental design:

• Isoform-specific antibodies: Select antibodies targeting epitopes unique to each isoform. Verify the epitope location relative to known splice junctions .

• Western blot optimization: Use gradient gels (4-15%) to achieve better separation of high-molecular-weight proteins. The canonical UNC13B has 1591 amino acids with a mass of 180.7 kDa .

• RT-PCR: Design primers spanning exon junctions specific to each isoform.

• Mass spectrometry: For definitive identification, use immunoprecipitation with UNC13B antibodies followed by mass spectrometry to identify isoform-specific peptides.

• Functional assays: Test cellular localization patterns, as isoforms may show differential distribution between membrane and cytoplasmic compartments .

Always confirm isoform identification through multiple complementary techniques, as antibody cross-reactivity between closely related isoforms can occur.

What are essential controls when working with UNC13B antibodies?

Implementing proper controls is critical for reliable UNC13B antibody experiments:

• Positive tissue/cell controls: Include samples known to express UNC13B (widely expressed across many tissue types) .

• Knockdown controls: Compare results between wildtype cells and those with UNC13B knockdown (shRNA/siRNA) .

• Peptide blocking: Pre-incubate antibody with immunizing peptide to confirm specificity.

• Isotype controls: Use non-specific IgG from the same host species as your primary antibody.

• Secondary-only controls: Omit primary antibody to detect non-specific secondary binding.

• Cross-reactivity assessment: Test antibody on samples from different species if working with non-human models.

Implementing these controls helps distinguish specific from non-specific signals and validates antibody performance in your experimental system.

How can I resolve non-specific binding issues with UNC13B antibodies?

When encountering non-specific binding with UNC13B antibodies, consider this troubleshooting approach:

• Increase blocking stringency: Test different blocking agents (BSA, normal serum, commercial blockers) and extend blocking time.

• Optimize antibody dilution: Test a broader dilution series than recommended (both higher and lower).

• Modify washing steps: Increase washing duration and number of washes between steps.

• Adjust buffer composition: Add detergents (0.1-0.3% Triton X-100 or Tween-20) to reduce hydrophobic interactions.

• Pre-adsorption: For problematic samples, pre-adsorb antibody with tissue powder or cell lysates from relevant negative control samples.

• Alternative antibody: Consider switching to antibodies targeting different epitopes of UNC13B .

Document all optimization steps methodically to establish a reproducible protocol for your specific experimental system.

How does UNC13B interact with mitochondrial pathways in cellular homeostasis?

UNC13B has emerged as a regulator of both apoptosis and mitochondrial fusion, particularly through its interaction with PINK1 (PTEN-induced putative kinase 1) . To investigate this:

• Co-immunoprecipitation: Use UNC13B antibodies to pull down protein complexes and probe for mitochondrial proteins like PINK1.

• Subcellular fractionation: Separate mitochondrial fractions and assess UNC13B localization during cellular stress.

• Live-cell imaging: Utilize fluorescently tagged UNC13B constructs alongside mitochondrial markers to visualize dynamic interactions.

• Functional assays: Measure mitochondrial membrane potential, ROS production, and mitophagy after UNC13B manipulation.

• Pathway analysis: Use Western blot to quantify changes in MAP3K7, CDK4, and PINK1 expression after UNC13B knockdown .

Research has shown that downregulation of UNC13B induces upregulation of PINK1, suggesting a regulatory relationship that may influence mitochondrial fusion dynamics and cellular response to stress conditions like drug exposure .

What methodological approaches can elucidate UNC13B's role in vesicle trafficking?

Given UNC13B's primary localization in vesicles and its role in promoting exocytosis through vesicle maturation , these approaches can help investigate its function:

• Total Internal Reflection Fluorescence (TIRF) microscopy: Monitor vesicle docking and fusion events in real-time using fluorescently tagged vesicle markers.

• Electrophysiology: Measure changes in exocytosis patterns after UNC13B manipulation in neuronal systems.

• Electron microscopy: Quantify vesicle distribution and morphology at the ultrastructural level.

• Proximity ligation assays: Detect protein-protein interactions between UNC13B and other components of the exocytic machinery.

• Calcium imaging: Assess the relationship between calcium signaling and UNC13B-mediated vesicle release.

• CRISPR domain editing: Generate specific mutations in functional domains to determine their relevance to vesicle trafficking.

These approaches can be complemented with immunostaining using UNC13B antibodies to correlate protein localization with functional outcomes.