FAX3 Antibody

What is Fas3 Antibody and what specific target does it recognize?

Fas3 Antibody (clone 7G10) is a monoclonal antibody that specifically recognizes Fasciclin III in Drosophila, a cell adhesion molecule critical for neural development. This antibody was developed using immunogen from membrane preparations of nerve cord and cultures of primary neurons collected during the 10-13 hour developmental period . The antibody is of the MIgG2a isotype and was produced using the NS-1 myeloma strain. Fas3 Antibody binds to the extracellular domain of the Fasciclin III protein, making it particularly useful for cell surface staining protocols that don't require detergent permeabilization .

What are the molecular characteristics of the Fasciclin III antigen?

Fasciclin III exists in multiple isoforms with apparent molecular weights of 46, 59, 66, and 80 kDa . This variation in molecular weight reflects different post-translational modifications and alternative splicing events that regulate the protein's function during development. The gene symbol is Fas3 with an Entrez Gene ID of 35097. While the complete antigen sequence is not provided in the available data, researchers should note that the epitope recognized by 7G10 is located on the extracellular domain of the protein, though precise epitope mapping has not been reported .

When was this antibody developed and who is the original depositor?

The Fas3 Antibody (7G10) was deposited to the Developmental Studies Hybridoma Bank (DSHB) on February 1, 1994, by Dr. C. Goodman from the University of California, Berkeley . This information can be important for proper citation in research publications and for understanding the historical context of reagents used in developmental neurobiology studies.

What are the recommended applications for Fas3 Antibody?

Based on depositor notes and published literature, Fas3 Antibody is particularly effective for:

• Immunohistochemistry of Drosophila tissue, especially for visualizing neurons and axons in the central nervous system

• Tracking specific neuronal subpopulations during development

• Affinity purification of Fasciclin III protein

There are conflicting reports regarding immunoprecipitation applications. While the depositor notes indicate that the antibody does not work for immunoprecipitation, the initial publication with the depositor as last author reports successful use of 7G10 for immunoprecipitation of Fasciclin III . This suggests that optimization of immunoprecipitation protocols may be necessary.

How should researchers prepare Drosophila samples for optimal Fas3 Antibody staining?

For optimal staining results with Fas3 Antibody, researchers should consider these methodological recommendations:

• For embryos beyond 10 hours of development, use dissected preparations rather than whole-mount staining, as ectodermal staining will obscure CNS signals in whole-mount preparations .

• The antibody can be used to stain dissections without detergent since the epitope is on the extracellular domain of the protein .

• For tracking specific neurons like RP1 and RP3, ensure proper orientation of dissected nervous system tissue to maintain anatomical context.

This methodological approach helps prevent false-negative results that might occur due to epitope masking by surrounding tissues during whole-mount staining procedures.

How can Fas3 Antibody be used to study neuronal circuit formation in Drosophila?

Fas3 Antibody is particularly valuable for studying neuronal circuit development because it specifically marks a subset of neurons and axons in the CNS . For advanced circuit mapping studies, researchers can combine Fas3 immunostaining with other neuronal markers to:

• Track the development of RP1 and RP3 neurons, for which this antibody is especially useful

• Analyze fasciculation patterns of specific axon bundles

• Investigate cell-cell interactions during neural circuit formation

When designing experiments to study circuit formation, it's important to consider developmental timing, as Fas3 expression changes throughout development. In larval stages, staining is present on a subset of neurons, the epithelial layer of the discs, and shows a distinctive "cyclone fence" pattern in the eye disc .

What techniques can be employed to distinguish between different isoforms of Fasciclin III?

Since Fasciclin III exists in multiple isoforms (46, 59, 66, and 80 kDa) , researchers might need to distinguish between these variants. While 7G10 antibody recognizes all isoforms, distinguishing between them requires additional techniques:

• Western blotting with high-resolution gels (though the depositor notes indicate limitations with Western blotting)

• Combining 7G10 with isoform-specific antibodies if available

• Using molecular approaches like RT-PCR to correlate isoform expression with antibody staining patterns

It's worth noting that many antibody-based applications for distinguishing protein isoforms benefit from technologies similar to those used in bispecific antibody development, where careful engineering can enhance specificity .

What are common technical challenges when using Fas3 Antibody and how can they be addressed?

Researchers working with Fas3 Antibody may encounter several technical challenges:

• CNS visualization in whole-mount preparations: After 10 hours of development, ectodermal staining obscures CNS signals. Solution: Use dissected preparations for embryos beyond this developmental stage .

• Immunoprecipitation inconsistencies: Conflicting reports exist regarding immunoprecipitation efficiency. Solution: Optimize buffer conditions and consider crosslinking approaches similar to those used in advanced antibody research platforms .

• Western blotting limitations: The depositor indicates Western blotting may not be effective. Solution: Consider alternative detection methods such as immunofluorescence or employ modern microfluidic antibody screening approaches for characterization .

How can researchers optimize tissue fixation and permeabilization protocols for Fas3 Antibody staining?

Since the Fas3 Antibody recognizes an epitope on the extracellular domain of the protein, fixation and permeabilization protocols require careful consideration:

• Membrane preservation: Use mild fixatives that preserve membrane structure while maintaining antigen accessibility.

• Detergent-free staining: For surface epitope visualization, protocols without detergent can be employed, similar to approaches used with live cells in antibody discovery platforms .

• Fixation timing: Optimize fixation duration to prevent over-fixation that might mask epitopes while ensuring sufficient tissue preservation.

For experiments requiring both surface and intracellular staining, sequential protocols can be developed where Fas3 surface staining is performed before permeabilization and subsequent intracellular antibody application.

How can Fas3 Antibody be used to study non-neuronal tissues in Drosophila?

While Fas3 Antibody is frequently used for neuronal studies, Fasciclin III expression extends to multiple non-neuronal tissues, offering additional research applications:

• Ectodermal development: Fas3 is expressed in patches of ectoderm and ectodermal stripes .

• Gut development: Parts of the hindgut and foregut express Fas3, allowing studies of gut tube formation .

• Visceral mesoderm: Fas3 expression in visceral mesoderm provides opportunities to study muscle development .

• Salivary gland morphogenesis: Fas3 expression in salivary glands enables studies of tubular organ formation .

These expression patterns make Fas3 Antibody a versatile tool for studying morphogenesis across multiple organ systems in Drosophila.

What controls should be included when using Fas3 Antibody in developmental studies?

For rigorous developmental studies using Fas3 Antibody, researchers should implement these controls:

• Negative controls: Include Fas3 mutant tissue or RNAi knockdown samples to confirm antibody specificity.

• Developmental series: Stain multiple developmental timepoints to establish expression dynamics.

• Co-staining controls: Use established markers for specific cell types to confirm cell identity.

Including these controls helps distinguish between specific and non-specific signals, particularly important when studying dynamic developmental processes.

How does the detection method influence Fas3 Antibody performance in different applications?

The choice of detection method can significantly impact experimental outcomes when using Fas3 Antibody. Consider these technical aspects:

• Signal amplification: For low-abundance epitopes, tyramide signal amplification or similar techniques may enhance detection sensitivity.

• Multiplex imaging: When combining Fas3 Antibody with other markers, select secondary antibodies with minimal spectral overlap.

• Live imaging considerations: For live applications utilizing the extracellular epitope, non-toxic fluorophore conjugates and minimally disruptive imaging protocols should be employed.

These considerations parallel advanced antibody screening methodologies that prioritize sensitivity and specificity in complex biological samples .

What are the comparative benefits of using Fas3 Antibody versus genetic reporters for tracking Fasciclin III expression?

This comparison helps researchers select the appropriate tool based on their specific experimental questions and available resources.

How might emerging antibody technologies enhance Fas3 research applications?

Recent advances in antibody technology could significantly expand Fas3 research capabilities:

• Bispecific antibody approaches: Technologies like SEED, ART-Ig, and Duobody platforms could be adapted to create reagents that simultaneously target Fas3 and other developmental markers.

• Microfluidic-enabled screening: High-throughput methods utilizing droplet microfluidics and FACS, as described for monoclonal antibody discovery , could be applied to develop new Fas3 antibodies with enhanced properties.

• Antibody fragment development: Generating Fab or scFv fragments from the original 7G10 clone could provide improved tissue penetration for thick specimens while maintaining specificity.

These technological adaptations could overcome current limitations and expand the research utility of Fas3 detection tools.