FAM160A2 Antibody

What is FAM160A2 and what cellular functions is it involved in?

FAM160A2, also known as FHIP (FTS and Hook-interacting protein), is a component of the FTS/Hook/FHIP complex (FHF complex). This protein plays a significant role in cellular trafficking mechanisms. The FHF complex functions primarily to promote vesicle trafficking and/or fusion via the homotypic vesicular protein sorting complex (the HOPS complex) . FAM160A2 has been identified as one of the FHIP family proteins that form specific interactions with Hook proteins, which are known adaptor proteins for cytoplasmic dynein-1 . Research indicates that FAM160A2 contributes to intracellular cargo transport pathways, making it an important target for studies focused on vesicular transport and organelle dynamics.

What applications are FAM160A2 antibodies validated for?

FAM160A2 antibodies have been validated for multiple research applications, with varying levels of validation depending on the specific antibody and manufacturer. Based on available scientific documentation, these antibodies are typically validated for:

Most commercially available FAM160A2 antibodies have been tested on human samples, with several manufacturers validating specificity through protein arrays containing the target protein alongside non-specific proteins . For optimal results, researchers should refer to product-specific datasheets as validation methods and recommended dilutions may vary between manufacturers.

What are the key differences between polyclonal and monoclonal FAM160A2 antibodies?

The choice between polyclonal and monoclonal FAM160A2 antibodies depends on the specific research needs and experimental design considerations.

Monoclonal FAM160A2 antibodies, like the EPR13604 clone from Abcam, are produced from a single B-cell clone and recognize a single epitope . These antibodies provide higher specificity and consistency between batches, making them valuable for studies requiring precise epitope targeting and reproducibility.

For complex protocols like co-immunoprecipitation studies involving FAM160A2 and its binding partners in the FHF complex, monoclonal antibodies may offer higher specificity, while polyclonal antibodies might provide better detection sensitivity in applications like Western blotting of complex cellular lysates.

How should FAM160A2 antibodies be stored to maintain optimal reactivity?

Proper storage of FAM160A2 antibodies is critical for maintaining their reactivity and extending their usable lifespan. Based on manufacturer recommendations:

For short-term storage (up to one month), FAM160A2 antibodies should be stored at 4°C . This temperature range minimizes antibody degradation while keeping the antibody in a ready-to-use state.

For long-term storage, aliquoting and storing at -20°C is recommended . Aliquoting helps prevent repeated freeze-thaw cycles, which can significantly damage antibody structure and function. Most commercial FAM160A2 antibodies are supplied in buffer solutions containing stabilizers such as:

• PBS, pH 7.2, containing 40% glycerol with 0.02% Sodium Azide

• 50% Glycerol, 0.01M PBS, pH 7.4 with preservatives like 0.03% Proclin 300

It is essential to avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of antibody activity. When working with conjugated antibodies (such as FITC-labeled FAM160A2 antibodies), additional precautions may be necessary, including protection from light to prevent photobleaching of the fluorophore .

What is the molecular structure and functional domains of FAM160A2?

FAM160A2 (UniProt ID: Q8N612) is a protein that functions as part of the FHF complex involved in vesicular trafficking. While detailed structural information from crystallography studies is limited in the current literature, functional analysis indicates that FAM160A2 contains specific domains that enable its interaction with Hook proteins and other components of the trafficking machinery .

The immunogen sequence used for antibody development often targets specific regions of the protein. For example, one antibody was developed against a recombinant protein corresponding to the amino acid sequence:
"ARQPLLRHGPVREALLTLLDACGRPVPSSPALDEGLVLLLSQLCVCVAQEPSLLEFFLQPPPEPGAAPRLLLFS"

This sequence likely represents a functionally important and/or antigenic region of the FAM160A2 protein. Research indicates that FAM160A2 interacts specifically with Hook1 and Hook3 proteins but not significantly with Hook2, suggesting domain-specific interaction capabilities . These interaction patterns differ from other FHIP family members (FHIP2A and FHIP2B), which display different Hook protein binding preferences.

How can researchers validate the specificity of FAM160A2 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For FAM160A2 antibodies, several validation approaches are recommended:

Positive Controls

• Use cell lines known to express FAM160A2 (information on expression levels in different cell lines can be found in antibody datasheets and expression databases)

• Recombinant FAM160A2 protein can serve as a positive control in Western blots

Negative Controls

• Use knockout or knockdown models where FAM160A2 expression is eliminated or reduced

• Include samples from tissues known not to express the target protein

Cross-Validation Methods

• Compare results using multiple antibodies targeting different epitopes of FAM160A2

• Validate findings using orthogonal methods (e.g., mass spectrometry)

Some manufacturers validate their FAM160A2 antibodies using protein arrays containing the target protein plus 383 other non-specific proteins to ensure specificity . This rigorous approach helps identify potential cross-reactivity issues before the antibody reaches researchers.

For applications like immunohistochemistry, pre-absorption tests with the immunizing peptide can help confirm specificity, where signal disappearance after pre-absorption indicates specific binding.

What are the known interactions between FAM160A2 and other proteins in the FHF complex?

FAM160A2 (also known as FHIP1B) is a component of the FTS/Hook/FHIP complex (FHF complex), with specific interaction patterns that have been characterized through various biochemical approaches. Recent research using proximity biotinylation and co-immunoprecipitation has revealed distinct interaction networks:

FAM160A2 (FHIP1B) preferentially interacts with Hook1 and Hook3, but not significantly with Hook2 . This interaction pattern distinguishes it from other FHIP family members like FHIP2A and FHIP2B, which show different Hook protein binding preferences.

The FHF complexes appear to form in specific combinations:

• FHIP1A/FHIP1B + Hook1/Hook3

• FHIP2A + Hook2/Hook3 (primarily Hook2)

• FHIP2B + Hook1/Hook2/Hook3

Coimmunoprecipitation experiments have confirmed these interaction patterns, with 3XFLAG-tagged FHIP1B expressed in 293T cells successfully coimmunoprecipitating Hook1 and Hook3, but not Hook2 . These specific interaction patterns suggest functional specialization among different FHF complexes, potentially relating to distinct cargo recognition or trafficking pathways.

What are the challenges in detecting different FHIP family members using commercial antibodies?

Detecting specific FHIP family members presents several challenges for researchers, particularly given the existence of multiple related proteins (FHIP1A, FHIP1B, FHIP2A, and FHIP2B) that may share sequence homology.

A significant challenge noted in the research literature is the variable quality of commercially available antibodies for different FHIP family members. For instance, researchers have reported that while antibodies for FHIP1B (FAM160A2) performed well in western blotting, commercially available antibodies for FHIP1A did not work satisfactorily in western blots . This limitation necessitated alternative approaches for confirming FHIP1A interactions.

Cross-reactivity between FHIP family members is another potential challenge, as these proteins share structural similarities. When selecting antibodies, researchers should:

• Verify epitope information to ensure the antibody targets unique regions that distinguish between family members

• Perform validation using overexpression systems of individual FHIP proteins

• Consider using antibodies raised against recombinant full-length proteins rather than peptide antibodies, which may increase specificity

For complex experiments involving multiple FHIP family members, using epitope-tagged versions of these proteins may provide a more reliable detection strategy than relying solely on commercially available antibodies.

What are the recommended protocols for immunoprecipitation using FAM160A2 antibodies?

Based on successful immunoprecipitation protocols reported in the literature, the following methodology is recommended for FAM160A2:

Sample Preparation

• Harvest cells and lyse in an appropriate lysis buffer (typically containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 or Triton X-100, with protease inhibitors)

• Centrifuge lysates at 14,000 × g for 10 minutes at 4°C to remove debris

• Determine protein concentration using a standard protein assay

Immunoprecipitation

• Pre-clear lysate with Protein G beads for 1 hour at 4°C

• Add 2-5 μg of FAM160A2 antibody to 500-1000 μg of pre-cleared lysate

• Incubate overnight at 4°C with gentle rotation

• Add 30-50 μl of Protein G beads and incubate for 2-4 hours at 4°C

• Wash beads 3-5 times with lysis buffer

• Elute proteins by boiling in SDS sample buffer

For studying interactions within the FHF complex, researchers have successfully used FLAG-tagged versions of FHIP proteins followed by immunoprecipitation with FLAG affinity resin . This approach can provide cleaner results when studying specific protein-protein interactions, especially when commercial antibodies show limitations.

How can researchers troubleshoot non-specific binding when using FAM160A2 antibodies in immunofluorescence?

Non-specific binding is a common challenge in immunofluorescence experiments. For FAM160A2 antibodies, consider the following troubleshooting strategies:

Optimize Blocking Conditions

• Test different blocking agents (BSA, normal serum, commercial blocking solutions)

• Increase blocking time (from 1 hour to overnight at 4°C)

• Add 0.1-0.3% Triton X-100 to blocking solution to improve antibody penetration

Antibody Dilution Optimization

• For FAM160A2 antibodies in immunofluorescence, the recommended range is typically 1-4 μg/mL

• Perform a dilution series to identify the optimal concentration that maximizes specific signal while minimizing background

Additional Controls

• Include a secondary antibody-only control to identify non-specific binding from the secondary antibody

• Use samples with known low or no expression of FAM160A2 as negative controls

• Pre-absorb the antibody with immunizing peptide to confirm specificity

Signal Amplification Considerations

When detecting low-abundance FAM160A2, consider:

• Using tyramide signal amplification systems

• Selecting brighter fluorophores (Alexa Fluor 488 or 568 rather than FITC)

• Extending primary antibody incubation time (overnight at 4°C)

FITC-conjugated FAM160A2 antibodies are available , which can eliminate secondary antibody-related background, but may require additional optimization steps due to potentially lower signal intensity compared to multi-step detection methods.

What is the role of FAM160A2 in vesicle trafficking pathways?

FAM160A2 functions as a component of the FTS/Hook/FHIP complex (FHF complex), which plays a crucial role in intracellular vesicle trafficking. Research indicates that this complex may function to promote vesicle trafficking and/or fusion via the homotypic vesicular protein sorting complex (the HOPS complex) .

Recent studies using proximity biotinylation approaches have revealed that different FHIP family members, including FAM160A2 (FHIP1B), interact with distinct sets of Hook proteins, suggesting specialized roles in cargo selection or trafficking pathways . Specifically, FAM160A2 interacts primarily with Hook1 and Hook3, which are known adaptor proteins for cytoplasmic dynein-1.

The FHF complex is believed to serve as a link between specific cargo and the dynein motor complex, facilitating minus-end directed movement along microtubules. Through these interactions, FAM160A2 likely contributes to:

• Endosomal trafficking and sorting

• Autophagosome transport

• Organelle positioning and dynamics

Understanding FAM160A2's role in these pathways has implications for research in cellular homeostasis, neurodegenerative diseases, and other conditions associated with defects in intracellular transport mechanisms.

What experimental considerations should researchers keep in mind when detecting FAM160A2 in different cellular compartments?

When investigating FAM160A2 localization and function in different cellular compartments, researchers should consider several experimental factors:

Fixation Methods

• For preserving membrane structures where FAM160A2 may associate, 4% paraformaldehyde is generally preferred over methanol

• For co-localization studies with cytoskeletal elements, combine paraformaldehyde with glutaraldehyde (0.1-0.5%)

Cell Types and Expression Levels

• Consider endogenous expression levels of FAM160A2 in different cell types when planning detection strategies

• For cells with low expression, signal amplification methods or more sensitive detection systems may be necessary

Co-localization Studies

• When studying FAM160A2's role in the FHF complex, co-staining with markers for Hook proteins can provide valuable information

• Consider using organelle-specific markers (early endosomes, late endosomes, lysosomes) to determine the specific trafficking pathways FAM160A2 is involved in

Technical Approaches

• For dynamic studies of FAM160A2 function, live cell imaging with fluorescently tagged proteins may be more informative than fixed cell approaches

• Super-resolution microscopy techniques can help resolve FAM160A2's precise localization within complex membrane systems

Controls for Specificity

• Include appropriate knockout or knockdown controls to verify antibody specificity in different cellular compartments

• Consider using multiple antibodies targeting different epitopes to confirm localization patterns

By carefully considering these experimental parameters, researchers can generate more reliable and informative data about FAM160A2's localization and function in cellular trafficking pathways.