Customize unc-94 Antibody

Description

Introduction to UNC-94 and Its Antibody

UNC-94 is a tropomodulin encoded by the unc-94 gene in C. elegans, essential for stabilizing actin filaments at sarcomere pointed ends and muscle cell boundaries . Polyclonal antibodies targeting UNC-94 were developed to investigate its localization, function, and interaction with cytoskeletal components. These antibodies have enabled discoveries in muscle sarcomere organization and epidermal morphogenesis .

Key Details:

Antigen: Residues 144–401 of UNC-94b (isoform-specific) .
Host: Rabbit-derived, affinity-purified using protein G .
Validation:
- Western Blot: Detects ~45 kDa bands (wild type), reduced signal in unc-94(su177) mutants, and no signal in unc-94(sf20) null mutants .
- Immunofluorescence: Localizes UNC-94 to sarcomeric thin filament ends and muscle cell boundaries .

Muscle Sarcomere Organization

Localization: UNC-94 antibodies revealed dual localization:
- Parallel lines flanking M-lines (thin filament pointed ends) .
- Accumulations near muscle cell-cell boundaries in mutants .
Phenotypic Analysis:
- unc-94(sf20) mutants show disorganized thin filaments and reduced motility .
- RNAi knockdown mimics mutant phenotypes, confirming specificity .

Epidermal Morphogenesis

Junctional Actin Stability: UNC-94 antibodies identified enrichment at seam cell boundaries, where it stabilizes actin networks during embryonic elongation .
Genetic Interaction: Synergistic defects in hmp-1(fe4);unc-94(RNAi) embryos highlight UNC-94’s role in α-catenin-dependent junctional integrity .

Table 2: Key Research Findings

Study Focus	Methodology	Major Findings
Sarcomere Assembly	IF, EM	UNC-94 prevents thin filament disorganization
Actin Dynamics	RNAi, Phalloidin	UNC-94 stabilizes junctional actin in epidermis
Isoform Expression	Promoter-GFP	Tissue-specific isoform expression (a vs. b)

Western Blotting

Sample Preparation: Extract proteins from synchronized worms.
Antibody Dilution: 1:1,000 with ECL detection .
Controls: Include unc-94(sf20) null mutants to confirm specificity .

Immunofluorescence

Fixation: Methanol-acetone fixation for muscle tissue .
Staining: Co-stain with phalloidin (F-actin) and DAPI (nuclei) .

Product Specs

Buffer

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

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

unc-94 antibody; tmd-1 antibody; C06A5.7 antibody; Tropomodulin antibody; Tmod-like protein antibody; cTmd1 antibody; Uncoordinated protein 94 antibody

Target Names

unc-94

Uniprot No.

O01479

Target Background

Function

UNC-94, also known as tropomodulin, serves as a pointed-end capping protein for actin filaments. This protein plays a crucial role in regulating the length and dynamic behavior of actin filaments by preventing both elongation and depolymerization at the pointed end.

Gene References Into Functions

Research suggests that UNC-94's minus-end actin capping function contributes to the proper formation and contraction of F-actin structures within the terminal web. This process is vital for maintaining the correct shape of the intestinal lumen. PMID: 24677443
In vitro studies demonstrate that UNC-94 collaborates with HMP-1 to produce longer actin bundles compared to HMP-1 alone. PMID: 22771044
The C-terminal portion of tropomodulin protein 1, isoform A, isolated from Caenorhabditis elegans, was successfully expressed in Escherichia coli and purified to homogeneity. PMID: 12777789
The structure of cTmd1, encompassing amino acids 196-392, has been determined. While the region from amino acids 222 to 388 is well-defined in the electron density map, the N-terminal segment (amino acids 196-221) is disordered. Similarly, the C-terminal tail (amino acids 389-392) exhibits some disorder. PMID: 15211521
Research has confirmed that the mutationally identified gene unc-94 corresponds to the predicted gene tmd-1 (C06A5.7), which encodes a tropomodulin. PMID: 17976644

Hide All

Database Links

KEGG: cel:CELE_C06A5.7

STRING: 6239.C06A5.7b

UniGene: Cel.17356

Protein Families

Tropomodulin family

Subcellular Location

Cytoplasm, cytoskeleton.

Q&A

The following FAQs address key research considerations for studying antibodies like UNC-94, synthesized from methodological insights in immunology research and antibody engineering. While specific UNC-94 data isn't available in public literature as of March 2025, these questions reflect common challenges in analogous antibody studies.

How to validate antibody specificity for UNC-94 in complex biological matrices?

Methodological approach:
- Perform cross-reactivity profiling using ELISA against related protein isoforms (e.g., paralogs with >80% sequence homology)
- Use immunoprecipitation followed by mass spectrometry to identify off-target binding partners
- Validate via CRISPR knock-out cell lines to confirm signal disappearance

Validation Strategy Comparison

Method	Sensitivity	Throughput	Cost
ELISA	High	Medium	$
Surface Plasmon Resonance	Ultra-high	Low	$$$
Flow Cytometry (Cell-based)	Medium	High	$$

What epitope mapping techniques are suitable for characterizing UNC-94's binding domain?

Advanced methodologies:
- Alanine scanning mutagenesis (resolution: 5-10Å)
- Cryo-EM for structural mapping of antibody-antigen complexes
- Deep mutational scanning to identify critical binding residues

How to resolve conflicting neutralization data between in vitro and in vivo models?

Contradiction analysis framework:
- Compare antibody affinity (KD) versus avidity measurements
- Test Fc receptor dependency using FcγR knockout models
- Analyze tissue penetration kinetics via PET-labeled antibodies

Case Study: Dengue Antibody Neutralization Variance

Genotype	Neutralization IC50 (μg/ml)	Structural Variation Site
Asian I	0.15	E-protein DIII K307R
American II	1.2	E-protein DI V52L

What computational strategies improve humanized antibody design for UNC-94?

Biophysical optimization pipeline:
- CDR grafting with RosettaAntibody framework analysis
- Molecular dynamics simulations (≥100ns trajectories) to assess stability
- Develop positional entropy scores for FR-CDR interfaces

Humanization Success Metrics

Parameter	Murine	Chimeric	Humanized
Immunogenicity Risk	High	Medium	<15%
Half-life (hr)	48	120	240

How to address batch-to-batch variability in recombinant UNC-94 production?

QC protocol enhancements:
- Implement HDX-MS for conformational stability monitoring
- Establish NMR fingerprinting for glycan heterogeneity ≤5%
- Use microfluidic SEC-MALS for aggregate quantification

What orthogonal assays confirm functional neutralization mechanisms?

Mechanistic validation suite:
- Biolayer interferometry for real-time binding kinetics (kon/koff)
- Neutrophil ADCC reporter assays (EC50 correlation ≥0.85)
- High-content imaging of target internalization

Key Methodological Considerations

Framework Region Optimization: Maintain ≥95% human consensus sequence in FR regions while preserving CDR structural integrity
Isotype Selection: IgG1 for complement activation vs IgG4 for reduced effector functions
Epitope Conservation Analysis: Use ancestral sequence reconstruction to predict escape variants

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unc-94 Antibody