UNC13B Antibody, HRP conjugated

What is UNC13B and why is it important in neurological research?

UNC13B (also known as Munc13-2) is a presynaptic protein that plays a crucial role in vesicle maturation during exocytosis as a target of the diacylglycerol second messenger pathway. It is involved in neurotransmitter release by facilitating synaptic vesicle priming prior to vesicle fusion and participates in the activity-dependent refilling of the readily releasable vesicle pool (RRP). UNC13B is essential for synaptic vesicle maturation particularly in excitatory/glutamatergic synapses, and in collaboration with UNC13A, it facilitates neuronal dense core vesicle fusion and controls both the location and efficiency of synaptic release . The protein's function in neurotransmitter release makes it a critical target for research into synaptic function, neurodevelopmental disorders, and neurodegenerative diseases.

What are the key applications for UNC13B antibodies in research?

UNC13B antibodies are valuable tools in neuroscience research with multiple applications:

• Western blotting (WB) for protein expression analysis

• Immunohistochemistry (IHC) for tissue localization

• Immunofluorescence (IF) for subcellular localization

• ELISA for quantitative analysis

• Flow cytometry (FACS) for cell analysis

• Co-immunoprecipitation for protein interaction studies

HRP-conjugated UNC13B antibodies are particularly useful for Western blotting and immunohistochemistry applications where direct detection without secondary antibodies is preferred . These applications allow researchers to investigate UNC13B's role in synaptic function, its interaction with other presynaptic proteins, and its distribution in different neuronal populations.

How does UNC13B differ from other UNC13 family members functionally?

UNC13B is one of several UNC13 isoforms with distinct functional characteristics. While both UNC13A and UNC13B contribute to synaptic vesicle priming, they exhibit differences in spatial distribution and functional roles:

• UNC13B is essential specifically for a subset of excitatory/glutamatergic synapses but not inhibitory/GABA-mediated synapses

• UNC13B works collaboratively with UNC13A in neuronal dense core vesicle fusion

• In Drosophila, the homologs Unc-13A and Unc-13B play different roles in release coupling at presynaptic active zones

These functional differences make isoform-specific antibodies critical for distinguishing the distinct roles of UNC13 family members in synaptic transmission.

What are the optimal conditions for using HRP-conjugated UNC13B antibodies in Western blotting?

For optimal Western blot results with HRP-conjugated UNC13B antibodies:

• Sample preparation:

  • Use fresh tissue or cell lysates extracted in RIPA buffer with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing Laemmli buffer

  • Load 10-30 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 5% SDS-PAGE gels due to UNC13B's high molecular weight (~180 kDa)

  • Transfer to PVDF membrane at low voltage (30V) overnight at 4°C for complete transfer

• Antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute HRP-conjugated UNC13B antibody at 1:500 to 1:10,000 depending on specific product

  • Incubate overnight at 4°C for best results

• Detection:

  • Wash extensively (3-5 times for 5-10 minutes each) with TBST

  • Develop using ECL substrate with exposure time optimized for signal intensity

  • For quantitative analysis, ensure signal falls within linear range

The advantage of HRP-conjugated antibodies is the elimination of secondary antibody incubation steps, which reduces background and cross-reactivity issues.

How should researchers optimize immunohistochemistry protocols for UNC13B detection?

For optimal immunohistochemistry results with UNC13B antibodies:

• Tissue preparation:

  • Fix tissues with 4% paraformaldehyde for 24-48 hours

  • Paraffin-embed or cryopreserve depending on experimental needs

  • Use 5-10 μm sections for optimal antibody penetration

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0)

  • Heat at 95-100°C for 20 minutes followed by 20-minute cooling

• Antibody dilution and incubation:

  • Block with serum-free protein block for 1 hour at room temperature

  • Dilute HRP-conjugated UNC13B antibody at 1:30 to 1:150 for IHC applications

  • Incubate overnight at 4°C in a humidified chamber

• Detection and counterstaining:

  • With HRP-conjugated antibodies, apply DAB substrate directly

  • Counterstain with hematoxylin for nuclear visualization

  • Mount with permanent mounting medium

For co-localization studies, consider multi-color fluorescence approaches with unconjugated primary antibodies and fluorophore-labeled secondary antibodies instead of HRP conjugates.

What controls should be included when using UNC13B antibodies in research?

Proper experimental controls are essential for validating UNC13B antibody results:

• Positive controls:

  • Brain tissue samples (particularly cerebral cortex or hippocampus)

  • Cell lines with confirmed UNC13B expression (e.g., neuronal cell lines)

  • Recombinant UNC13B protein as positive control for Western blotting

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues known to lack UNC13B expression

  • Knockdown or knockout samples where available

• Specificity controls:

  • Pre-absorption with immunizing peptide

  • Comparison with alternative antibodies targeting different epitopes of UNC13B

  • Correlation of results across multiple detection methods

• Loading/staining controls:

  • Housekeeping proteins (β-actin, GAPDH) for Western blotting

  • DAPI nuclear staining for immunofluorescence

These controls help ensure that observed signals are specific to UNC13B rather than artifacts or non-specific binding.

What are common issues when using HRP-conjugated UNC13B antibodies and how can they be resolved?

Common challenges with HRP-conjugated UNC13B antibodies include:

• High background in Western blots:

  • Increase blocking duration (2-3 hours)

  • Reduce antibody concentration

  • Add 0.1-0.3% Tween-20 to washing buffer

  • Increase number and duration of washes

  • Use specialized blocking agents (e.g., BSA, commercial blockers)

• Weak or absent signal:

  • Increase protein loading (30-50 μg)

  • Reduce washing stringency

  • Optimize antibody concentration

  • Confirm sample preparation preserves epitope integrity

  • Verify protein transfer efficiency with reversible stain

  • Check ECL substrate freshness and activity

• Multiple bands or unexpected band sizes:

  • UNC13B may undergo post-translational modifications or alternative splicing

  • Verify specificity with knockout/knockdown controls

  • Use reducing conditions consistently

  • Prevent proteolytic degradation with additional protease inhibitors

  • Run longer SDS-PAGE (8-10 cm) for better resolution of high molecular weight proteins

• Irreproducible results:

  • Standardize lysate preparation methods

  • Use consistent antibody lot numbers

  • Maintain detailed protocols with all parameters

  • Store antibody according to manufacturer recommendations

For HRP-conjugated antibodies specifically, ensure proper storage conditions to maintain enzymatic activity and avoid freeze-thaw cycles.

How can researchers quantitatively analyze UNC13B expression in different experimental conditions?

For rigorous quantitative analysis of UNC13B expression:

• Western blot densitometry:

  • Capture images within linear detection range

  • Normalize UNC13B band intensity to loading controls

  • Use three or more biological replicates

  • Apply statistical testing appropriate for experimental design

  • Report fold-change with standard error

• ELISA quantification:

  • Develop standard curve with recombinant UNC13B

  • Use HRP-conjugated UNC13B antibody at 1:5000-1:10000 dilution

  • Ensure samples fall within linear range of standard curve

  • Run all samples in technical triplicates

• Immunofluorescence quantification:

  • Use consistent acquisition parameters

  • Measure integrated density or mean fluorescence intensity

  • Analyze multiple fields (>5) per sample

  • Normalize to area or cell number

  • Use automated analysis workflows to reduce bias

• Flow cytometry (FACS):

  • Gate populations consistently across samples

  • Calculate mean fluorescence intensity

  • Compare to isotype controls

  • Use compensation when multiple fluorophores are present

For all quantitation methods, ensure appropriate statistical analysis and transparent reporting of all analytical parameters.

How should researchers interpret conflicting results between different UNC13B antibodies?

When facing discrepancies between different UNC13B antibodies:

• Consider epitope differences:

  • Map the specific epitopes recognized by each antibody (e.g., AA 12-215 vs. AA 1062-1091)

  • Different domains may be differentially accessible in various experimental conditions

  • Some epitopes may be masked by protein-protein interactions

• Evaluate antibody validation:

  • Review validation data provided by manufacturers

  • Check literature for independent validation

  • Conduct knockout/knockdown validation if possible

• Assess technical variables:

  • Different applications (WB vs. IHC) have different requirements

  • Some antibodies work better in native vs. denatured conditions

  • Different fixation methods may affect epitope accessibility

• Integration approach:

  • Prioritize results from antibodies with stronger validation

  • Use multiple antibodies targeting different epitopes

  • Consider orthogonal methods (mRNA analysis, tagged protein expression)

  • Report discrepancies transparently in publications

When possible, use functionally validated antibodies that have been shown to detect changes in known physiological or pathological conditions affecting UNC13B.

How can UNC13B antibodies be utilized in super-resolution imaging studies?

UNC13B antibodies can be powerful tools for super-resolution microscopy investigations:

• Sample preparation considerations:

  • For STORM/PALM: Use primary UNC13B antibodies with secondary antibodies conjugated to photoswitchable fluorophores

  • For STED: Use bright, photostable fluorophores

  • For SIM: Standard immunofluorescence protocols are generally sufficient

  • For best results, use thin sections (≤10 μm) and optimize fixation

• Co-localization studies:

  • UNC13B can be co-localized with active zone proteins like Brp/ELKS

  • Multi-color STORM allows precise mapping of protein relationships

  • As demonstrated in Drosophila studies, super-resolution imaging reveals nanoscale reorganization of Unc-13 at presynaptic active zones

• Analysis approaches:

  • HDBSCAN cluster analysis for precise determination of UNC13B nanoarrangement

  • Measure radial distance parameters to quantify spatial organization

  • Calculate co-localization coefficients with other synaptic proteins

• Functional correlations:

  • Combine with electrophysiology to correlate structure with function

  • Track changes in UNC13B distribution during synaptic plasticity

  • Analyze reorganization during homeostatic challenges

Super-resolution approaches have revealed that UNC13 undergoes nanoscale reorganization during presynaptic homeostatic potentiation, with decreased radial distance and smaller extent of the UNC13 area per active zone .

What are the considerations for using UNC13B antibodies in studies of neurodegenerative diseases?

When investigating UNC13B in neurodegenerative disorders:

• Disease-specific considerations:

  • UNC13 family proteins have been implicated in various neurological disorders

  • Changes in expression or localization may correlate with disease progression

  • Post-translational modifications may be disease-specific

• Tissue handling:

  • Post-mortem interval affects protein integrity

  • Disease-specific fixation protocols may be necessary

  • Consider laser-capture microdissection for region-specific analysis

• Analytical approaches:

  • Compare UNC13B levels between affected and unaffected brain regions

  • Correlate UNC13B changes with markers of synaptic loss

  • Assess co-localization with disease-specific protein aggregates

  • Examine relationship with other presynaptic proteins

• Methodological recommendations:

  • Use multiple antibodies targeting different UNC13B epitopes

  • Include age-matched controls

  • Account for medication effects in human samples

  • Consider cellular resolution techniques (single-cell RNA-seq with protein validation)

• Functional correlation:

  • Link UNC13B changes to electrophysiological measurements

  • Correlate with behavioral deficits in animal models

  • Assess relationship to synaptic vesicle parameters

How can researchers use UNC13B antibodies to study activity-dependent synaptic reorganization?

For investigating activity-dependent changes in UNC13B:

• Experimental paradigms:

  • Pharmacological manipulation (PhTx treatment for homeostatic potentiation)

  • Optogenetic stimulation protocols

  • Learning paradigms in animal models

  • Long-term potentiation (LTP) or depression (LTD) protocols

• Analytical approaches:

  • Super-resolution imaging to track nanoscale reorganization

  • Measure UNC13B cluster size, density, and distribution

  • Track radial distance parameters during plasticity

  • Quantify co-localization with calcium channels or other active zone proteins

• Temporal considerations:

  • Acute versus chronic adaptations

  • Time-course studies to capture dynamic reorganization

  • Activity-dependent changes in UNC13B mobility

• Combined methodologies:

  • Correlate structural changes with electrophysiological measurements

  • Pair with calcium imaging to link with presynaptic calcium dynamics

  • Combine with synaptic vesicle labeling to assess functional impact

Studies using super-resolution microscopy have demonstrated that UNC13 undergoes compaction at the active zone during acute homeostatic challenges, potentially reflecting functional compensation for enhanced neurotransmitter release . These structural changes, while subtle in 2D localization data, may translate to significant changes in 3D molecular configuration.

What is the optimal approach for investigating UNC13B interactions with other presynaptic proteins?

For studying UNC13B protein interactions:

• Co-immunoprecipitation strategy:

  • Use unconjugated UNC13B antibodies for immunoprecipitation

  • Mild lysis conditions to preserve protein complexes (e.g., 1% NP-40)

  • Pre-clear lysates to reduce non-specific binding

  • Include appropriate controls (IgG, knockout samples)

  • Confirm results with reciprocal IP using antibodies to interaction partners

• Proximity ligation assay (PLA):

  • Use pairs of antibodies (UNC13B + interaction partner)

  • Optimize antibody concentrations to minimize background

  • Include controls (single antibody, unrelated protein pairs)

  • Quantify PLA puncta per defined cellular area

• FRET/BiFC approaches:

  • For recombinant systems examining direct interactions

  • Design constructs to avoid interfering with interaction domains

  • Include positive and negative interaction controls

  • Validate findings with endogenous proteins

• Super-resolution co-localization:

  • Particularly valuable for studying relationships with active zone proteins

  • Future studies investigating co-localization of UNC13 with RBP, VGCCs, or RIM would be informative

  • Use analytical approaches that account for resolution limitations

These approaches provide complementary information about the spatial organization and direct interaction of UNC13B with other presynaptic components.

What are the considerations for studying UNC13B in different model systems?

When investigating UNC13B across different experimental models:

How should researchers interpret the relationship between UNC13B protein levels and functional synaptic changes?

For meaningful interpretation of UNC13B expression and function:

• Expression vs. localization:

  • Total UNC13B levels (Western blot/ELISA) may not reflect functional changes

  • Subcellular distribution (immunostaining) may be more functionally relevant

  • Nanoscale organization at active zones correlates with function

• Post-translational modifications:

  • Phosphorylation state affects function independently of total protein

  • Consider phospho-specific antibodies when available

  • Functional state may depend on association with lipid messengers

• Relationship to synaptic vesicle parameters:

  • Correlate UNC13B changes with readily releasable pool (RRP) size

  • Measure synaptic vesicle priming using electrophysiology

  • Assess relationship to release probability

• Causal relationships:

  • Acute manipulation (optogenetics, pharmacology) helps establish causality

  • Genetic approaches (conditional knockout) provide temporal control

  • Rescue experiments confirm specificity

• Quantitative frameworks:

  • Develop mathematical models linking UNC13B levels to vesicle fusion

  • Consider non-linear relationships between expression and function

  • Account for compensatory mechanisms and redundancy with other UNC13 isoforms

Understanding that moderate structural changes in UNC13B localization may translate to larger functional effects is important for interpretation .