SLC31A2 Antibody

What is SLC31A2 and why is it important in copper transport research?

SLC31A2, also known as copper transporter 2 (CTR2), is a key player in copper transport and regulation within the body. It belongs to the Solute Carrier Family 31 and is crucial for maintaining copper homeostasis in cells. SLC31A2 is a multi-pass membrane protein primarily localized to late endosomes, lysosomes, and cytoplasmic vesicles, where it acts upstream of or within cellular copper ion homeostasis pathways . In humans, the canonical protein has a reported length of 143 amino acid residues and a mass of approximately 15.7 kDa .

The importance of studying SLC31A2 stems from its role in copper metabolism, which when dysregulated, has been linked to several disorders including Menkes disease, Wilson's disease, and certain types of cancer. Understanding the function and regulation of SLC31A2 provides insights into copper-related pathologies and potential therapeutic approaches .

What types of SLC31A2 antibodies are available for research purposes?

Several types of SLC31A2 antibodies are available for research purposes, each with specific characteristics and applications:

The rabbit polyclonal antibody is generated against a synthetic peptide corresponding to a sequence within amino acids 1-100 of mouse SLC31A2 (NP_001277447.1) . The mouse monoclonal antibodies target specific peptide epitopes of human SLC31A2 and have been validated through various techniques including immunohistochemistry and immunofluorescence .

How do you validate the specificity of an SLC31A2 antibody?

Validating the specificity of an SLC31A2 antibody requires a multi-faceted approach to ensure reliable results:

• Positive Control Testing: Use tissues or cell lines known to express SLC31A2, such as mouse pancreas for mouse-reactive antibodies . The Human Protein Atlas provides validation data for human-reactive antibodies, including positive staining in THP-1 cells .

• Western Blot Analysis: Confirm the antibody detects a protein of the expected molecular weight (approximately 16 kDa for SLC31A2) . Observe for single, clean bands at the expected size.

• Peptide Competition Assays: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific binding should be blocked by the peptide.

• Multiple Application Testing: Validate across different applications (WB, ELISA, IHC, IF) to ensure consistent results. For example, CPTC-SLC31A2-1 shows positive results in both IHC and IF applications .

• Cross-Reactivity Assessment: Test the antibody against related proteins (like SLC31A1/CTR1) to ensure specificity to SLC31A2.

• Knockout/Knockdown Controls: Where possible, use SLC31A2 knockout or knockdown samples as negative controls.

Validation results from organizations like the Human Protein Atlas provide valuable reference data to assess antibody quality before use in experimental settings .

What are the recommended dilutions and applications for SLC31A2 antibodies?

The optimal dilutions and applications for SLC31A2 antibodies vary based on the specific antibody and experimental context:

For immunofluorescence applications, the subcellular localization observed with CPTC-SLC31A2-1 is supported by the literature, with staining primarily in the plasma membrane of human cell lines . When introducing a new antibody into your research, it's advisable to perform a dilution series to determine the optimal concentration for your specific experimental conditions.

How do different fixation and permeabilization methods affect SLC31A2 antibody performance?

Fixation and permeabilization methods significantly impact SLC31A2 antibody performance due to the protein's membrane localization and potential conformational sensitivity:

Fixation Effects:

Paraformaldehyde (PFA) fixation has been successfully used for SLC31A2 detection in immunofluorescence applications, as demonstrated with CPTC-SLC31A2-1 on THP-1 and U2OS cell lines . This cross-linking fixative preserves membrane structure while maintaining epitope accessibility.

Optimization Considerations:

• Membrane Protein Considerations: As a multi-pass membrane protein, SLC31A2 requires careful optimization of permeabilization to balance between preserving membrane integrity and allowing antibody access.

• Fixative Concentration and Time: Lower PFA concentrations (2-4%) with shorter fixation times may better preserve epitopes compared to stronger fixation conditions.

• Permeabilization Agents: For membrane proteins like SLC31A2, milder detergents (0.1% Triton X-100 or 0.1-0.3% saponin) may be preferable to stronger agents that can disrupt membrane structure.

• Antigen Retrieval: For paraffin-embedded tissues, citrate or EDTA-based antigen retrieval methods may help expose SLC31A2 epitopes without damaging tissue architecture.

Researchers should consider comparing multiple fixation methods when establishing SLC31A2 detection protocols, particularly when studying its distribution across different cellular compartments (plasma membrane vs. endosomal/lysosomal locations).

What are the considerations for using SLC31A2 antibodies to investigate copper-related disorders?

Using SLC31A2 antibodies to investigate copper-related disorders requires careful experimental design and consideration of disease-specific contexts:

Experimental Design Considerations:

• Tissue Selection: Focus on tissues with high SLC31A2 expression or those affected in copper-related disorders (liver, brain, intestine, kidney). Mouse pancreas has been identified as a positive sample for SLC31A2 detection .

• Disease Context: Consider how SLC31A2 expression, localization, or function might be altered in specific copper disorders:

  • Menkes disease: Focus on impaired copper delivery to critical enzymes

  • Wilson's disease: Examine copper accumulation and potential compensatory mechanisms

  • Cancer studies: Investigate altered copper metabolism in malignant transformation

• Antibody Selection: Choose antibodies validated in the relevant species and tissues. For human studies, monoclonal antibodies like CPTC-SLC31A2-1 with positive Human Protein Atlas validation offer reliable detection .

Technical Approach:

When investigating potential therapeutic targets, researchers should consider how SLC31A2 interacts with other copper transporters and regulators, as dysregulation of SLC31A2 has been linked to copper-related disorders including Menkes disease, Wilson's disease, and certain types of cancer where copper metabolism is dysregulated .

What are the best practices for Western blot analysis of SLC31A2?

Western blot analysis of SLC31A2 requires specific considerations due to its relatively small size (16 kDa) and membrane localization:

Optimized Western Blot Protocol:

• Sample Preparation:

  • Use specialized lysis buffers containing 1% NP-40 or Triton X-100 with protease inhibitors

  • Include gentle sonication to aid membrane protein solubilization

  • Avoid boiling samples (heat to 37°C for 30 minutes instead)

• Gel Electrophoresis:

  • Use higher percentage gels (15-18%) for better resolution of low molecular weight proteins

  • Load appropriate positive controls (mouse pancreas for mouse-reactive antibodies)

• Transfer Conditions:

  • Use PVDF membranes (0.2 μm pore size) for small proteins

  • Consider semi-dry transfer systems with 20% methanol buffer

• Antibody Application:

  • For the rabbit polyclonal antibody (CAB16362), use dilutions between 1:500 and 1:2000

  • Include overnight incubation at 4°C for optimal binding

• Detection:

  • Use high-sensitivity chemiluminescent substrates

  • Consider longer exposure times for detecting low abundance signals

Expected Results:

The calculated and observed molecular weight for SLC31A2 is approximately 16 kDa . Clean Western blots should show a single band at this molecular weight, though post-translational modifications like ubiquitination may result in additional higher molecular weight bands .

What is the potential for developing antibodies with engineered specificity profiles for SLC31A2 research?

The development of antibodies with engineered specificity profiles represents a frontier in SLC31A2 research, offering unprecedented control over targeting specific epitopes or variants:

Advanced Engineering Approaches:

Recent work demonstrates the feasibility of designing antibodies with customized specificity profiles through a combination of experimental selection and computational modeling . For SLC31A2 research, this approach enables:

• Epitope-Specific Targeting:

  • Design antibodies that selectively recognize specific domains of SLC31A2

  • Create reagents that distinguish between closely related copper transporters (SLC31A1/CTR1 vs. SLC31A2/CTR2)

• Conformation-Selective Antibodies:

  • Engineer antibodies that recognize specific conformational states associated with copper transport activity

  • Develop tools to distinguish between active and inactive forms of the transporter

• Cross-Species Optimized Reagents:

  • Design antibodies with controlled cross-reactivity across model organisms

  • Enable comparative studies across species with consistent detection tools

Implementation Strategy:

The biophysics-informed modeling approach involves training on experimentally selected antibodies and associating distinct binding modes with potential ligands . This enables:

• Predictive Applications: Using data from one ligand combination to predict outcomes for others

• Generative Capabilities: Creating antibody variants not present in initial libraries that specifically target desired epitopes

For SLC31A2, this could mean developing antibodies that distinguish between the protein in different cellular compartments or functional states, providing more nuanced tools for copper transport research.

What controls should be included when using SLC31A2 antibodies in immunoprecipitation studies?

Immunoprecipitation (IP) studies with SLC31A2 antibodies require rigorous controls to ensure valid and reproducible results:

Essential IP Controls:

• Input Control:

  • Purpose: Confirms presence of SLC31A2 in starting material

  • Implementation: Reserve 5-10% of pre-IP lysate for parallel Western blot analysis

• Negative Controls:

  • Purpose: Establishes background binding level

  • Types:

    • Isotype control (IgG from same species as SLC31A2 antibody)

    • Pre-immune serum (for polyclonal antibodies)

    • IP from SLC31A2-negative cell lines

• Specificity Controls:

  • Purpose: Confirms antibody specifically recognizes SLC31A2

  • Implementation: Peptide competition assay using the immunizing peptide

  • For CAB16362: Use the synthetic peptide corresponding to amino acids 1-100 of mouse SLC31A2

• Technical Controls:

  • No-antibody beads control (beads only)

  • Reverse IP validation (IP with antibodies against suspected interaction partners)

IP Protocol Considerations:

For membrane proteins like SLC31A2, special considerations include:

• Use mild detergents (0.5-1% NP-40 or digitonin) to preserve protein-protein interactions

• Include protease and phosphatase inhibitors freshly in all buffers

• Consider crosslinking antibodies to beads to prevent antibody contamination in eluates

• For analysis of ubiquitination , include deubiquitinase inhibitors in lysis buffers

A comprehensive IP experiment should incorporate these controls to distinguish genuine interactions from technical artifacts when studying SLC31A2 biology.

How might advanced antibody engineering techniques enhance SLC31A2 research in the coming years?

Advanced antibody engineering techniques are poised to significantly enhance SLC31A2 research through several transformative approaches:

Emerging Technologies and Applications:

• Single-Domain Antibodies (Nanobodies):

  • Smaller size allows better access to sterically hindered epitopes

  • Potential for detecting SLC31A2 in native membrane environments

  • Applications in super-resolution microscopy to map precise subcellular localization

• Bispecific Antibodies:

  • Simultaneous targeting of SLC31A2 and interaction partners

  • Enables visualization of transient copper transport complexes

  • Applications in studying the interplay between multiple copper transporters

• Antibody-Based Biosensors:

  • Development of conformation-sensitive antibodies to detect active vs. inactive states

  • Creation of FRET-based reporters for real-time monitoring of copper transport

  • Integration with optogenetic tools for spatiotemporal control of SLC31A2 function

Computational Design Approaches:

The biophysics-informed modeling approach demonstrated for antibody development offers particular promise for SLC31A2 research:

• Epitope-Focused Libraries: Design of antibody libraries specifically targeting functionally important domains of SLC31A2

• Cross-Reactivity Engineering: Development of antibodies with precisely controlled reactivity across species and SLC31 family members

• Structure-Based Optimization: As structural information about SLC31A2 increases, computational approaches can design antibodies targeting specific conformational states

These advanced antibody engineering techniques will enable more precise dissection of SLC31A2 biology and its role in copper homeostasis, potentially leading to novel therapeutic strategies for copper-related disorders .

What are the prospects for using SLC31A2 antibodies in developing therapeutics for copper-related disorders?

The use of SLC31A2 antibodies in developing therapeutics for copper-related disorders represents an exciting frontier with several potential approaches:

Therapeutic Development Pathways:

• Diagnostic Applications:

  • Development of antibody-based assays to detect altered SLC31A2 expression/localization in patient samples

  • Creation of companion diagnostics to identify patients likely to respond to copper-modulating therapies

  • Implementation of immunohistochemistry panels to characterize copper transport profiles in disease states

• Targeted Delivery Systems:

  • Engineering antibody-drug conjugates targeting cells with aberrant SLC31A2 expression

  • Development of nanoparticle delivery systems guided by anti-SLC31A2 antibodies

  • Creation of bispecific antibodies linking SLC31A2-expressing cells to immune effectors

• Functional Modulation:

  • Design of antibodies that can modulate SLC31A2 transport activity

  • Development of antibody-based approaches to restore proper localization in mislocalization disorders

  • Creation of intrabodies to influence SLC31A2 trafficking within cells

Research to Clinical Translation:

The path from current research tools to therapeutic applications will require several advances:

• Humanized Antibodies: Engineering of fully human or humanized antibodies against SLC31A2 to minimize immunogenicity

• Tissue-Specific Targeting: Development of delivery systems that can target SLC31A2 in specific tissues affected in different copper disorders

• Functional Characterization: Deeper understanding of how SLC31A2 dysregulation contributes to specific disease mechanisms

The established link between SLC31A2 dysregulation and copper-related disorders like Menkes disease, Wilson's disease, and certain cancers provides a strong foundation for these therapeutic development efforts.