SLC39A10 Antibody

What is SLC39A10 and what are its key biological functions?

SLC39A10 (Solute Carrier Family 39 Member 10), also known as ZIP10 or KIAA1265, is a zinc-influx transporter protein with a calculated molecular weight of approximately 94 kDa (94132 Da) . It belongs to the SLC39 family of zinc transporters and functions primarily to facilitate zinc uptake into cells .

The protein demonstrates several critical biological functions:

• Acts as a zinc-influx transporter across cellular membranes

• Forms functional heterodimers with SLC39A6, which mediate cellular zinc uptake to trigger epithelial-to-mesenchymal transition (EMT)

• Plays an essential role in initiating mitosis by importing zinc into cells

• Contributes to mature B-cell maintenance and humoral immune responses

• Regulates NCAM1 phosphorylation and integration into focal adhesion complexes during EMT

SLC39A10 is primarily localized to the cell membrane as a multi-pass membrane protein and is notably expressed at the apical membranes of proximal tubules in the kidney . Its expression has been documented across multiple species including human, mouse, and rat tissues .

What applications are SLC39A10 antibodies suitable for?

SLC39A10 antibodies have been validated for multiple research applications with specific methodological considerations for each technique:

When selecting an antibody for your specific application, consider the target epitope location (N-terminal, C-terminal, or central regions) as this may affect binding efficacy in different experimental contexts . Validation using positive controls (such as cell lysates with known SLC39A10 expression) is essential before proceeding with experimental samples .

How should SLC39A10 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of SLC39A10 antibodies are crucial for maintaining their specificity and sensitivity:

Storage recommendations:

• Short-term storage (up to three months): 4°C

• Long-term storage (up to one year): -20°C

• Avoid repeated freeze-thaw cycles, which can significantly compromise antibody performance

• Do not expose antibodies to prolonged high temperatures

When reconstituting lyophilized antibodies, use sterile techniques and follow manufacturer-specific protocols. After reconstitution, aliquot the antibody into small volumes to minimize freeze-thaw cycles if long-term storage is needed .

For experimental use, it's advisable to prepare fresh working dilutions on the day of the experiment. When diluting, use appropriate buffers (typically PBS with 0.1% BSA or manufacturer-recommended diluents) to maintain antibody stability .

What controls should be included when using SLC39A10 antibodies in experimental protocols?

Including appropriate controls is essential for validating SLC39A10 antibody specificity and experimental outcomes:

Essential controls:

• Positive control: Cell lysates or tissues with confirmed SLC39A10 expression

• Negative control: Samples from knockout models or cell lines with confirmed absence of SLC39A10

• Isotype control: Non-specific IgG from the same host species at equivalent concentration

• Secondary antibody-only control: To assess background signal

• Blocking peptide control: Using the immunizing peptide to confirm specificity

For Western blot applications, confirm that the detected band appears at the expected molecular weight (~94 kDa) . When interpreting results, note that SLC39A10 antibodies do not cross-react with the related transporter ZIP11, allowing for specific detection .

For immunohistochemistry, include tissue sections known to express SLC39A10 (such as kidney proximal tubules) as anatomical positive controls .

What is the relationship between SLC39A10 and cancer progression?

Recent research has revealed significant connections between SLC39A10 and cancer development, particularly in gastric cancer:

SLC39A10 has been identified as a functional oncogene in gastric cancer, with several mechanistic pathways implicated:

• Expression patterns: SLC39A10 is frequently upregulated in gastric adenocarcinomas, and this upregulation strongly correlates with poor patient outcomes

• Regulation mechanism: SLC39A10 has been identified as a direct target of the c-Myc oncogene

• Functional effects: Ectopic expression of SLC39A10 in gastric cancer cells significantly enhances:

  • Cellular proliferation

  • Colony formation

  • Invasiveness

  • Tumorigenic potential in nude mice

Mechanistically, SLC39A10 exerts its oncogenic effects through:

• Increasing zinc (Zn²⁺) availability within cancer cells

• Enhancing the enzymatic activity of casein kinase 2 (CK2)

• Activating downstream MAPK/ERK and PI3K/AKT signaling pathways

• Establishing a vicious feedback loop with c-Myc to drive malignant progression

These findings suggest that targeting CK2 could represent an alternative therapeutic strategy for gastric cancer patients with high SLC39A10 expression . Research examining SLC39A10 expression in tumors may benefit from using antibodies that specifically target epitopes that remain accessible in the cancer microenvironment.

How can researchers measure and modulate SLC39A10 activity in cellular systems?

Assessing SLC39A10 functional activity requires methodologies beyond simple protein detection:

Zinc uptake measurement approaches:

• Radiolabeled zinc (⁶⁵Zn) uptake assays: LLC-PK₁ cells expressing rZip10 demonstrated time-, temperature-, and concentration-dependent zinc uptake with saturable kinetics (Km of 19.2 μM, Vmax of 50 pmol·min⁻¹·mg protein⁻¹)

• Fluorescent zinc indicators: FluoZin-3 or Zinquin can be used to measure intracellular zinc levels

• ICP-MS (Inductively Coupled Plasma Mass Spectrometry): For precise quantification of total cellular zinc content

SLC39A10 activity modulation:

• Competitive inhibition: Cadmium can competitively inhibit rZip10-mediated zinc uptake with a Ki of 91 μM

• Chemical modification: COOH group-modifying agents such as DCC (dicyclohexylcarbodiimide) inhibit rZip10-mediated zinc transport

• Expression regulation: SLC39A10 expression is responsive to extracellular zinc levels, showing upregulation at moderate zinc concentrations but downregulation at higher concentrations

For experimental designs studying SLC39A10 function, establishing stable cell lines expressing SLC39A10 (such as LLC-PK₁-Zip10 cells) provides a controlled system for functional analyses . When conducting zinc uptake experiments, researchers should carefully control for background zinc uptake through endogenous transporters.

What methodological approaches can differentiate SLC39A10 from other zinc transporters?

Distinguishing SLC39A10 from other zinc transporters requires specific experimental design considerations:

Antibody selection strategies:

• Epitope specificity: Select antibodies targeting unique regions of SLC39A10 that are not conserved across other ZIP family members

• Cross-reactivity testing: SLC39A10 antibodies specifically do not cross-react with the related transporter ZIP11

• Specificity validation: Use synthetic peptides corresponding to different regions of SLC39A10 for competition assays

Expression analysis approaches:

• RT-PCR with SLC39A10-specific primers: Can be used to detect Slc39a10 mRNA levels

• RNA interference: siRNA or shRNA targeting unique regions of SLC39A10 mRNA

• Immunoprecipitation: Using SLC39A10-specific antibodies to pull down the protein and associated complexes

When studying SLC39A10 function in heterodimer formation with SLC39A6, co-immunoprecipitation assays using antibodies against both proteins can help elucidate their interaction dynamics . Additionally, subcellular localization studies using immunofluorescence can distinguish SLC39A10 from other ZIP family members based on its characteristic localization patterns, such as its expression at the apical membranes of kidney proximal tubules .

How do post-translational modifications affect SLC39A10 detection using antibodies?

Post-translational modifications (PTMs) of SLC39A10 can significantly impact antibody detection:

Key considerations:

• Epitope accessibility: PTMs may mask antibody binding sites, particularly for antibodies targeting regions prone to modification

• Conformation changes: Phosphorylation or other modifications may alter protein folding, affecting antibody recognition

• Processing events: Proteolytic cleavage may remove epitopes or generate fragments with altered molecular weights

When working with SLC39A10 antibodies, researchers should consider:

• Selecting antibodies targeting multiple regions (N-terminal, central, C-terminal) to ensure detection regardless of modification status

• Including phosphatase inhibitors in lysis buffers when studying potential phosphorylation events, particularly when examining SLC39A10's role in signaling pathways

• Using denaturing vs. native conditions to account for conformation-dependent epitope recognition

• Comparing detection patterns across multiple techniques (WB, IP, IF) to comprehensively assess SLC39A10 status

How can researchers optimize Western blot protocols for SLC39A10 detection?

Western blot optimization for SLC39A10 requires attention to several technical parameters:

Recommended protocol adjustments:

• Protein extraction: Use RIPA buffer supplemented with protease inhibitors and zinc chelators (if studying zinc-dependent interactions)

• Gel percentage: 8-10% SDS-PAGE gels are optimal for resolving the 94 kDa SLC39A10 protein

• Transfer conditions: Extended transfer times (overnight at low voltage) may improve transfer efficiency of this large membrane protein

• Blocking: 5% non-fat dry milk in TBST is generally effective, but BSA may be preferable if phospho-specific detection is needed

• Antibody concentration: Start with 1-2 μg/mL and optimize based on signal-to-noise ratio

• Incubation conditions: Overnight at 4°C typically yields best results for primary antibody

• Detection method: HRP-conjugated secondary antibodies with enhanced chemiluminescence provide good sensitivity

Common issues and solutions:

• Multiple bands: May indicate proteolytic degradation (add more protease inhibitors) or different isoforms

• No signal: Check protein loading, transfer efficiency, and antibody working concentration

• High background: Increase washing steps, optimize blocking conditions, or try a different antibody dilution

For particularly challenging samples, membrane fractionation to enrich for membrane proteins may improve SLC39A10 detection, as it is primarily localized to the plasma membrane .

What are the key considerations for immunohistochemical detection of SLC39A10?

For successful immunohistochemical staining of SLC39A10 in tissue sections:

Critical protocol elements:

• Fixation: 10% neutral buffered formalin is standard, but fixation time should be optimized (excessive fixation may mask epitopes)

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically required for formalin-fixed tissues

• Antibody concentration: Begin at 2.5 μg/mL and titrate as needed

• Incubation time: Overnight at 4°C often yields optimal staining

• Detection system: ABC (avidin-biotin complex) or polymer-based detection systems provide good sensitivity

• Counterstaining: Hematoxylin provides good nuclear contrast

Tissue-specific considerations:

• Kidney sections: Focus on proximal tubules where SLC39A10 is prominently expressed at apical membranes

• Cancer tissues: May show altered expression patterns compared to normal tissues

• Control tissues: Include known positive tissues in each staining run

When interpreting staining patterns, remember that SLC39A10 should primarily show membrane localization, with potential additional cytoplasmic staining depending on cellular context . Comparison with other zinc transporter staining patterns can help confirm specificity.

How can researchers address non-specific binding when using SLC39A10 antibodies?

Non-specific binding can complicate SLC39A10 detection but can be minimized through several approaches:

Optimization strategies:

• Antibody dilution: Titrate to find optimal concentration that maximizes specific signal while minimizing background

• Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers) and concentrations

• Washing stringency: Increase number and duration of wash steps with detergent-containing buffers

• Secondary antibody selection: Choose highly cross-adsorbed secondary antibodies to reduce non-specific binding

• Pre-adsorption: For immunohistochemistry, pre-incubate antibody with acetone powder of non-relevant tissues

Validation approaches:

• Peptide competition: Pre-incubate antibody with the immunizing peptide to confirm specificity of staining

• Knockout/knockdown controls: Compare staining in samples with confirmed absence of SLC39A10

• Multiple antibodies: Use antibodies targeting different epitopes of SLC39A10 to confirm staining patterns

How is SLC39A10 being studied in relation to zinc homeostasis and disease pathways?

Recent research has expanded our understanding of SLC39A10's role in zinc regulation and pathological conditions:

Zinc homeostasis mechanisms:

• SLC39A10 expression is dynamically regulated by extracellular zinc levels, with initial upregulation at moderate zinc concentrations followed by downregulation at higher concentrations

• The transporter demonstrates saturable zinc uptake kinetics following Michaelis-Menten parameters (Km of 19.2 μM)

• SLC39A10-SLC39A6 heterodimers have emerged as key regulators of cellular zinc import for specific physiological processes

Disease-related research:

• Cancer progression: SLC39A10 upregulation promotes gastric cancer through enhanced zinc availability and subsequent CK2 activation

• Signaling pathway interactions: SLC39A10 activates MAPK/ERK and PI3K/AKT pathways in cancer cells

• Oncogenic feedback loops: SLC39A10 forms a feedback regulatory circuit with c-Myc to drive malignant progression

These findings suggest SLC39A10 could represent a potential therapeutic target, particularly in cancers with aberrant zinc metabolism . Research utilizing antibodies against various epitopes of SLC39A10 has been instrumental in elucidating these mechanisms by enabling detection of SLC39A10 interactions, modifications, and localization patterns.

What considerations should researchers make when comparing data from different SLC39A10 antibodies?

When integrating data generated using different SLC39A10 antibodies, researchers should consider several factors:

Critical comparison parameters:

• Epitope differences: Antibodies targeting different regions (N-terminal, central, C-terminal) may yield different results based on epitope accessibility or post-translational modifications

• Clonality variations: Polyclonal antibodies may recognize multiple epitopes, while monoclonal antibodies target a single epitope

• Host species considerations: Different host species (rabbit, mouse) may yield antibodies with varying affinities and specificities

• Validation methods: Consider how each antibody was validated (Western blot, immunohistochemistry, knockout controls)

Data integration strategies:

• Cross-validation: Use multiple antibodies targeting different epitopes to confirm findings

• Application-specific optimization: An antibody that works well for Western blot may not be optimal for immunoprecipitation

• Standardized protocols: When comparing results across studies, consider differences in experimental protocols

For comprehensive SLC39A10 research, using a panel of antibodies recognizing different epitopes can provide more complete information about protein expression, localization, and potential modifications. This is particularly important when studying SLC39A10 in disease contexts where processing or interactions may be altered .