Customize snb-1 Antibody

Description

SNB-1 Protein and Antibody Overview

SNB-1 is a neuronal v-SNARE protein essential for synaptic vesicle fusion and neurotransmitter release . The SNB-1 antibody (designated SB1) was developed to study synaptic architecture and vesicle dynamics. It recognizes an epitope (PRPSNKRLQQ) within the N-terminal cytoplasmic domain (amino acids 1–86) of SNB-1 .

Antibody Development and Validation

Immunogen: Mice were immunized with a His₆-tagged SNB-1 fusion protein lacking the transmembrane domain .
Hybridoma lines: Three IgG1 clones (2E6, 5D1, 5G1) were generated, with 5D1 (SB1) showing superior specificity and deposited in the Developmental Studies Hybridoma Bank (DSHB) .
Specificity:
- Western blots detect a 16 kDa band in wild-type lysates, shifted to 19 kDa in snb-1(md247) mutants .
- Immunostaining localizes SNB-1 to synaptic vesicles in the nerve ring, dorsal/ventral nerve cords, and presynaptic terminals .

Synaptic Vesicle Localization

SB1 enables visualization of synaptic vesicle distribution in GABAergic motor neurons. Key findings include:

Reduced anterograde vesicle flux in casy-1 mutants, rescued by CASY-1C overexpression .
Impaired vesicle mobility after unc-104 RNAi, highlighting UNC-104/KIF1A's role in transport .

Protein Interaction Studies

Co-immunoprecipitation: SB1 confirmed SNB-1 interactions with UNC-10 (active zones) and SAD-1 kinase (neuronal polarity) .
Colocalization: SNB-1 puncta overlap with presynaptic markers like UNC-10 and SNT-1 .

Mutant Characterization

snb-1 mutants (md247, js17, js124) exhibit aldicarb resistance and synaptic transmission deficits .

Table 1: SNB-1 Antibody Characteristics

Parameter	SB1 Performance
Target Gene	snb-1
Subcellular Localization	Synaptic vesicles
Western Blot	+++ (16 kDa band in WT)
Immunohistochemistry	+++ (nerve ring, cords)
Epitope	PRPSNKRLQQ (cytoplasmic)

Key Research Findings

Neuronal Polarity: SNB-1 interacts with SAD-1 kinase and Neurabin (NAB-1) to restrict axonal fate .
Vesicle Transport: CASY-1 C-terminal recruits UNC-104/KIF1A to regulate GABAergic vesicle motility .
Synaptic Defects: snb-1 mutants show aberrant vesicle clustering and neurotransmitter release .

Technical Considerations

Limitations: SB1 does not recognize the transmembrane domain, limiting use in full-length SNB-1 studies .
Alternatives: GFP-tagged SNB-1 (hpIs66) enables live imaging of vesicle dynamics .

Product Specs

Buffer

Preservative: 0.03% ProClin 300; Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

14-16 weeks (Made-to-order)

Synonyms

snb-1 antibody; T10H9.4 antibody; Synaptobrevin-1 antibody; Synaptobrevin-related protein 1 antibody

Target Names

snb-1

Uniprot No.

O02495

Target Background

Function

This antibody targets a protein involved in the targeting and fusion of transport vesicles with their target membranes. It plays a role in neuronal exocytosis during synaptic transmission, likely contributing to cholinergic transmission. This protein is essential for cell viability, coordinated movement, and the proper function of M3 pharynx motor neurons.

Database Links

KEGG: cel:CELE_T10H9.4

STRING: 6239.T10H9.4

UniGene: Cel.5604

Protein Families

Synaptobrevin family

Subcellular Location

Cytoplasmic vesicle, secretory vesicle, synaptic vesicle membrane; Single-pass type IV membrane protein. Cell membrane. Cell junction, synapse, synaptosome.

Tissue Specificity

Expressed in the nervous system notably the nerve ring, ventral cord and dorsal cord.

Q&A

Basic Research Questions

How is the SNB-1 antibody validated for synaptic vesicle localization studies in C. elegans?
- Methodology:
  - Use immunofluorescence on whole-mount C. elegans preparations. Compare staining patterns in wild-type and snb-1(md247) mutants. A 16 kDa band (wild-type) shifting to 19 kDa in mutants confirms specificity via Western blot .
  - Include controls like pre-immune serum and secondary antibody-only samples to rule out nonspecific binding.
- Key Data:
  
  Assay Wild-Type Signal Mutant Signal Citation
  Western Blot 16 kDa band 19 kDa (reduced intensity)
  Immunohistochemistry Nerve ring, dorsal/ventral cords Absent in mutants
What experimental designs optimize SNB-1 antibody use in dual-labeling studies?
- Recommendations:
  - Pair monoclonal SNB-1 antibody (e.g., SB1, IgG1 isotype ) with polyclonal antibodies targeting other synaptic markers (e.g., UNC-10/RIM for active zones).
  - Use sequential staining with intermediate fixation steps to avoid cross-reactivity.

Assay	Wild-Type Signal	Mutant Signal	Citation
Western Blot	16 kDa band	19 kDa (reduced intensity)
Immunohistochemistry	Nerve ring, dorsal/ventral cords	Absent in mutants

Advanced Research Questions

How do SNB-1 antibody-epitope interactions influence mechanistic studies of synaptic trafficking?
- Epitope Mapping:
  - The SB1 antibody binds the SNB-1 N-terminal sequence PRPSNKRLQQ . Design truncation mutants to test epitope necessity for vesicle docking.
  - Combine cryo-EM with antibody labeling to visualize SNB-1 spatial organization in synaptic vesicles.
How to resolve contradictions in SNB-1 antibody reactivity across model systems?
- Troubleshooting Framework:
  - Cross-reactivity: Test antibody on heterologous systems (e.g., HEK293 cells expressing SNB-1 homologs ).
  - Post-translational modifications: Treat lysates with phosphatases/kinases to assess phosphorylation-dependent epitope masking .

Methodological Challenges

What quantitative approaches improve SNB-1 antibody-based synaptic vesicle density measurements?
- Advanced Imaging:
  - Apply Airyscan super-resolution microscopy to resolve clustered SNB-1 punctae (<200 nm spacing).
  - Use Fiji/ImageJ macros for automated puncta counting, normalized to axonal length (e.g., Puncta Analyzer plugin).
How does SNB-1 antibody performance compare to CRISPR-tagged endogenous SNB-1 reporters?
- Validation Strategy:
  
  Metric Antibody-Based CRISPR Tagging
  Temporal resolution Limited by fixation Real-time tracking
  Artifact risk Epitope occlusion Tag interference
  - Validate findings with both methods in parallel .

Metric	Antibody-Based	CRISPR Tagging
Temporal resolution	Limited by fixation	Real-time tracking
Artifact risk	Epitope occlusion	Tag interference

Data Interpretation Guidelines

False Positives: Confirm SNB-1 knockdown via qPCR before attributing signal loss to antibody failure.
Context-Dependent Variability: Account for developmental stage (e.g., L4 vs. adult worms show 2.3-fold signal difference ).

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snb-1 Antibody