slc49a3 Antibody

What is SLC49A3 and why is it significant for research?

SLC49A3 (solute carrier family 49 member 3) is a protein-coding gene located on chromosome 4p16.3. It is predicted to enable transmembrane transporter activity and is thought to be involved in transmembrane transport . The protein is predicted to be active in the membrane, though its precise function remains to be fully characterized .

SLC49A3 is also known as MFSD7 and belongs to the SLC49 family, which includes other members like FLVCR1 (SLC49A1), FLVCR2 (SLC49A2), and DIRC2 (SLC49A4) . While some members of this family have been implicated in specific diseases (e.g., FLVCR1 in posterior column ataxia with retinitis pigmentosa and FLVCR2 in Fowler syndrome), the specific functions of SLC49A3 remain largely unknown . The protein has gained research interest due to its potential role in transport mechanisms and its involvement in certain fusion proteins associated with sarcomas .

What is known about the structure and expression pattern of SLC49A3?

SLC49A3 is encoded by a gene with 15 exons on chromosome 4 . According to genomic data, the gene sequence spans from position 676826 to 691624 on the complement strand of chromosome 4 (NC_000004.12) .

While detailed expression data isn't provided in the available search results, SLC49A3 (as MFSD7) has been identified as a fusion partner with ATP5I in certain human sarcomas, suggesting expression in connective tissues . The MFSD7-ATP5I fusion transcript was detected in 58% of sarcoma samples in one study and was associated with increased cell migration and invasion properties .

As a membrane protein, SLC49A3 is predicted to contain multiple transmembrane domains characteristic of the major facilitator superfamily, though detailed structural studies would be needed to confirm its topology.

What approaches can be used to generate effective antibodies against SLC49A3?

While the search results don't specifically detail methods for SLC49A3 antibody generation, we can learn from approaches used for similar proteins. Based on methodologies used for generating antibodies against aminoacyl-tRNA synthetases (aaRSs) , the following approaches would be applicable:

• Recombinant Antigen Production: Express specific domains or the full-length SLC49A3 protein in bacterial systems (like E. coli) with appropriate tags (e.g., Avi-tag for site-specific biotinylation) .

• Phage Display Selection: Use synthetic human single-chain fragment variable (scFv) libraries for selection against the purified SLC49A3 protein .

• Domain-Specific Targeting: Since SLC49A3 is a membrane protein with multiple domains, targeting specific extracellular or cytoplasmic domains might yield antibodies with different applications (detection vs. functional blocking).

• Hybridoma Technology: As an alternative to phage display, traditional hybridoma approaches could be used to generate monoclonal antibodies against purified SLC49A3 protein or peptides derived from its sequence.

For recombinant antibody production, the protein would typically be purified using a two-step procedure involving immobilized metal affinity chromatography (IMAC) followed by size-exclusion chromatography (SEC) to ensure high purity and monodispersity .

How should SLC49A3 antibodies be validated for research applications?

Based on the validation approaches used for other antibodies , a comprehensive validation workflow for SLC49A3 antibodies should include:

• Primary Binding Assays: ELISA and homogeneous time-resolved fluorescence (HTRF) to confirm binding to the purified antigen .

• Specificity Testing: Suspension bead assays (e.g., Luminex) to test for cross-reactivity against related proteins, particularly other members of the SLC49 family .

• Immunoprecipitation-Mass Spectrometry (IP-MS): This gold standard validation determines whether the antibody can capture endogenous SLC49A3 from cell lysates. Successful antibodies would show the highest normalized spectral abundance factor (NSAF) for SLC49A3 .

• Immunofluorescence: To confirm the ability of the antibody to detect the native protein in fixed cells and to verify its subcellular localization .

• Western Blotting: To confirm specificity for SLC49A3 at the expected molecular weight.

• Knockout/Knockdown Controls: Using CRISPR/Cas9 knockout or siRNA knockdown cells to confirm antibody specificity.

For antibodies intended for therapeutic or diagnostic use, additional validation including tissue cross-reactivity studies would be essential.

How can SLC49A3 antibodies be used to study protein-protein interactions?

SLC49A3 antibodies can be powerful tools for studying protein-protein interactions through several approaches:

• Co-Immunoprecipitation (Co-IP): SLC49A3 antibodies could be used to pull down the protein along with its interaction partners from cell lysates. This approach has been successfully used with antibodies against members of the multi-tRNA synthetase complex (MSC) to identify interaction networks .

• Proximity Labeling: SLC49A3 antibodies could be combined with techniques like BioID or APEX2 proximity labeling to identify proteins in close proximity to SLC49A3 in living cells.

• Cross-linking Mass Spectrometry: Using antibodies to purify SLC49A3 after chemical cross-linking could help identify transient or weak interaction partners.

• Immunofluorescence Co-localization: Dual immunofluorescence staining with SLC49A3 antibodies and antibodies against potential interaction partners can provide evidence for co-localization in cellular compartments.

Given that SLC49A3 has been found as part of a fusion protein (MFSD7-ATP5I) in sarcomas , antibodies could potentially be used to study how this fusion affects normal protein interactions and contributes to pathogenesis.

What techniques can be employed to investigate SLC49A3 localization and trafficking?

To study SLC49A3 localization and trafficking, researchers could employ:

• Immunofluorescence Microscopy: Using validated SLC49A3 antibodies for fixed-cell imaging to determine subcellular localization .

• Live-Cell Imaging: Though not directly using antibodies, the information from antibody validation could inform the design of fluorescent protein fusions for dynamic tracking of SLC49A3.

• Subcellular Fractionation: Combined with Western blotting using SLC49A3 antibodies to biochemically determine the protein's distribution across cellular compartments.

• Immunoelectron Microscopy: For high-resolution localization studies, particularly important for membrane proteins like SLC49A3.

• Surface Biotinylation Assays: To specifically study the plasma membrane population of SLC49A3 and its internalization kinetics.

Since SLC49A3 is predicted to be a transmembrane transporter , understanding its localization is crucial for elucidating its function. If it follows patterns similar to other SLC transporters, it might show specialized localization to particular membrane compartments or cell types.

How can SLC49A3 antibodies be used to investigate its potential role in cancer?

Given the association of the MFSD7-ATP5I fusion with sarcomas and the implication of related family member DIRC2 (SLC49A4) in hereditary renal carcinomas , SLC49A3 antibodies could be valuable for cancer research:

• Tissue Microarray Analysis: SLC49A3 antibodies could be used for immunohistochemistry on cancer tissue microarrays to assess expression patterns across different cancer types and stages.

• Fusion Protein Detection: Developing antibodies that specifically recognize the MFSD7-ATP5I fusion junction could help diagnose sarcomas where this fusion is present. The fusion has been detected in 58% of sarcoma samples and is associated with increased cell migration and invasion .

• Functional Studies: Using blocking antibodies against SLC49A3 in cell-based assays could help determine if inhibiting the protein affects cancer cell proliferation, migration, or invasion.

• Biomarker Development: Similar to how SLC46A3 has been investigated as a biomarker for antibody-drug conjugates , SLC49A3 could potentially serve as a diagnostic or prognostic marker if its expression correlates with disease states.

Research has shown that MFSD7-ATP5I expression is associated with marked pleomorphism and lower tumor necrosis in sarcomas, and experiments have demonstrated that this fusion promotes invasiveness of tumor cells through the GSK-3 pathway .

What are the common challenges when working with SLC49A3 antibodies and how can they be addressed?

Based on general challenges with membrane protein antibodies and information from similar research , likely challenges include:

• Low Endogenous Expression: If SLC49A3 is expressed at low levels, detection may be difficult. Solution: Use enrichment techniques like immunoprecipitation before detection or employ signal amplification methods in immunostaining.

• Accessibility of Epitopes: Membrane proteins often have limited exposed regions for antibody binding. Solution: Generate antibodies against multiple domains and use membrane permeabilization methods optimized for transmembrane proteins.

• Cross-reactivity with Related Proteins: The SLC49 family contains several members with potential structural similarities . Solution: Extensive specificity testing against all family members (SLC49A1, SLC49A2, SLC49A4) is essential, as demonstrated in validation studies for other protein families .

• Fixation Sensitivity: Some epitopes may be destroyed by certain fixation methods. Solution: Compare multiple fixation protocols (PFA, methanol, acetone) to determine optimal conditions for immunofluorescence.

• Batch-to-batch Variability: This is particularly problematic with polyclonal antibodies. Solution: Use recombinant antibody technology as described for other targets , where sequences can be deposited in public databases to ensure reproducibility.

How can antibodies help determine the substrate specificity of SLC49A3?

As SLC49A3 is predicted to function as a transmembrane transporter , determining its substrates is crucial:

• Blocking Studies: Antibodies targeting extracellular domains could potentially block transport activity if accessible, allowing researchers to study the functional consequences in cellular assays.

• Transport Assays: While not directly using antibodies, insights from antibody-based localization studies can inform the design of transport assays using radioisotope or fluorescently labeled potential substrates.

• Co-localization with Known Transporters: Antibodies could be used to determine if SLC49A3 co-localizes with other transporters of known specificity, providing clues about potential substrates.

• Pull-down of Transported Molecules: In some cases, antibodies can be used to immunoprecipitate the transporter along with bound substrates for identification by mass spectrometry.

Given that other members of the SLC49 family like FLVCR1 and FLVCR2 have been linked to heme transport , investigating whether SLC49A3 might also transport heme or related compounds would be a logical direction.

What approaches can be used to study post-translational modifications of SLC49A3 using antibodies?

Post-translational modifications (PTMs) often regulate transporter function and localization. To study PTMs of SLC49A3:

• Modification-specific Antibodies: Develop antibodies that specifically recognize phosphorylated, glycosylated, or ubiquitinated forms of SLC49A3.

• Two-dimensional Immunoblotting: Use general SLC49A3 antibodies with 2D gel electrophoresis to separate different post-translationally modified forms of the protein.

• Immunoprecipitation-Mass Spectrometry: Use SLC49A3 antibodies to pull down the endogenous protein, followed by mass spectrometry analysis to identify PTMs .

• Stimulus Response Studies: Examine changes in PTMs after cellular stimulation (e.g., stress, growth factors) by immunoprecipitating SLC49A3 from treated and untreated cells.

• Site-directed Mutagenesis Validation: Use SLC49A3 antibodies to compare the behavior of wild-type vs. mutant proteins where potential PTM sites have been altered.

Understanding PTMs could provide insights into how SLC49A3 function is regulated and how it might contribute to pathological conditions when dysregulated.