slc30a5 Antibody

What is SLC30A5 and why is it important to study?

SLC30A5 (also known as ZnT5) belongs to the SLC30A family of zinc transporters that mediate zinc efflux from the cytoplasm to either the extracellular space or intracellular vesicles. It plays a critical role in zinc homeostasis, which is essential for numerous cellular processes including gene expression, enzyme activity, cell proliferation, and apoptosis. Recent studies have shown that SLC30A5 is upregulated in various cancers, particularly hepatocellular carcinoma (HCC), and its expression correlates with adverse prognosis, advanced disease stages, higher histological grades, and vascular invasion . The protein has emerged as a promising candidate for both a prognostic marker and a potential therapeutic target, making antibodies against SLC30A5 valuable research tools for investigating its roles in normal physiology and disease.

What are the recommended validation methods for SLC30A5 antibodies?

When validating SLC30A5 antibodies for research applications, several complementary approaches should be employed:

• Positive and negative controls: Use cell lines or tissues known to express high levels of SLC30A5 as positive controls. For negative controls, consider using tissues with low expression or cells with SLC30A5 knockdown.

• Western blot validation: SLC30A5 protein typically appears as bands at approximately 75 kDa (monomeric form) and sometimes at ~150 kDa (possibly representing dimeric forms or post-translationally modified variants) . Comparison with the migration pattern observed in previous studies can help confirm antibody specificity.

• Genetic knockdown validation: Generate cells with SLC30A5 knockdown using shRNA or CRISPR-Cas9 approaches. The specific shRNA sequence used in published research (5'-CCGGCCAGATAATTGGATCACTAAACTCGAGTTTAGTGATCCAATTATCTGGTTTTTG-3') has been validated for SLC30A5 targeting in Huh7 cells .

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody specifically recognizes SLC30A5 rather than other related proteins.

• Cross-reactivity testing: Evaluate potential cross-reactivity with other SLC30A family members, particularly those with high sequence homology.

What experimental methods are best suited for SLC30A5 protein detection?

Multiple techniques can be used to detect SLC30A5 protein, each with specific advantages:

• Western blotting: Effective for quantitative analysis of SLC30A5 expression levels. Based on published research, SLC30A5 is detected as bands at approximately 75 kDa (monomeric form) and sometimes at ~150 kDa . Use appropriate lysis buffers containing zinc chelators to preserve protein integrity.

• Immunohistochemistry (IHC): Useful for examining SLC30A5 expression patterns in tissue sections. Paraffin-embedded tissue sections with antigen retrieval techniques have been successfully employed in cancer studies.

• Immunofluorescence: Valuable for subcellular localization studies, as SLC30A5 has been reported to localize to both plasma membrane and intracellular vesicular structures .

• ELISA: Commercial ELISA kits are available for quantitative measurement of SLC30A5 in various sample types .

• Flow cytometry: Can be used to quantify SLC30A5 expression in cell populations, particularly when examining expression in specific cell subsets.

How does SLC30A5 expression vary across normal and cancerous tissues?

SLC30A5 expression shows distinct patterns between normal and cancerous tissues:

Normal tissues:

• Moderate expression in most tissues with zinc-dependent functions

• Higher baseline expression in pancreatic tissue

• Expression in immune cells such as macrophages, with potential regulation during immune responses

Cancer tissues:

• Significantly overexpressed in various tumors, particularly hepatocellular carcinoma (HCC)

• Notable expression in malignant cells as confirmed by single-cell RNA sequencing data (GSE112271)

• Elevated expression observed in tumor cell lines derived from the central nervous system (CNS), lung, and liver

• Moderate to high expression in pancreatic cancer

The differential expression pattern makes SLC30A5 a potential biomarker for cancer detection and prognosis. Research indicates that in HCC, upregulation of SLC30A5 correlates with adverse prognosis, advanced disease stages, higher histological grades, and vascular invasion .

What protocols can optimize SLC30A5 detection in Western blotting?

For optimal detection of SLC30A5 by Western blotting, consider the following protocol refinements:

• Protein extraction: Use a lysis buffer containing protease inhibitors and zinc chelators to prevent protein degradation. RIPA buffer supplemented with 1 mM PMSF, 1 mM sodium orthovanadate, and a protease inhibitor cocktail has been effective in previous studies.

• Denaturation conditions: Heat samples at 95°C for 5 minutes in Laemmli buffer containing 5% β-mercaptoethanol.

• Gel percentage: Use 8-10% SDS-PAGE gels for optimal separation of the 75 kDa and 150 kDa SLC30A5 forms.

• Transfer conditions: Transfer to PVDF membranes (preferred over nitrocellulose for higher binding capacity) at 100V for 90 minutes in cold transfer buffer containing 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody dilution: Typically, primary SLC30A5 antibodies are used at 1:1000 dilution in 5% BSA in TBST, with overnight incubation at 4°C.

• Detection: Both chemiluminescence and fluorescence-based detection systems are suitable, with the latter offering better quantification capabilities.

• Special considerations: Note that SLC30A5 may exhibit different molecular weights due to post-translational modifications, particularly after stimulation with factors like LPS in immune cells .

What is the subcellular localization of SLC30A5 and how can it be visualized?

SLC30A5 exhibits a dynamic subcellular localization that varies by cell type and activation state:

• General localization: SLC30A5 can localize to both the plasma membrane and intracellular vesicular structures, as demonstrated in studies of human macrophages .

• Visualization techniques:

  • Confocal microscopy with immunofluorescence staining is the gold standard for visualizing SLC30A5 subcellular localization

  • Co-localization studies with organelle markers (e.g., LAMP1 for lysosomes, GM130 for Golgi, Calnexin for ER) can precisely identify SLC30A5-containing compartments

  • Live-cell imaging using GFP-tagged SLC30A5 can track dynamic localization changes

• Protocol optimization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 3% BSA for 30 minutes

  • Incubate with primary antibody (typically 1:100-1:200) overnight at 4°C

  • Use appropriate fluorophore-conjugated secondary antibodies (1:500-1:1000)

  • Include DAPI or Hoechst for nuclear counterstaining

• Functional localization: In macrophages responding to bacterial infection, SLC30A family proteins can relocalize to facilitate zinc delivery to bacteria-containing compartments as part of an antimicrobial response .

How can I design experiments to study SLC30A5's role in zinc transport and cellular function?

To investigate SLC30A5's role in zinc transport and cellular function, consider these experimental approaches:

• Genetic manipulation strategies:

  • shRNA knockdown: Use validated shRNA sequences (e.g., 5'-CCGGCCAGATAATTGGATCACTAAACTCGAGTTTAGTGATCCAATTATCTGGTTTTTG-3') to reduce SLC30A5 expression in cell lines like Huh7

  • CRISPR-Cas9 knockout: Generate complete knockout cell lines for more dramatic phenotypes

  • Overexpression systems: Use doxycycline-inducible expression systems similar to those employed for other SLC30A family members

• Zinc transport assays:

  • Fluorescent zinc probes: Use FluoZin-3 or Zinpyr-1 to measure intracellular free zinc levels

  • Radioactive zinc (65Zn) uptake/efflux assays: Quantify zinc transport rates in control versus SLC30A5-modified cells

  • Zinc-sensitive reporter systems: Employ bacterial zinc-responsive promoters fused to reporter genes (similar to the MG1655 zinc stress reporter system used for SLC30A1 studies)

• Functional assays:

  • Cell proliferation: Use CCK8 viability assay and EdU cell proliferation assay to assess growth effects

  • Colony formation: Evaluate clonogenic potential in control versus SLC30A5-knockdown cells

  • Apoptosis assays: Measure cell death using Annexin V/PI staining and flow cytometry

  • Migration and invasion: Use wound healing and transwell migration assays to assess metastatic potential

• In vivo models:

  • Xenograft studies: Employ mouse models with cells modified for SLC30A5 expression to evaluate tumor growth and progression in vivo

  • Zinc supplementation/depletion: Combine SLC30A5 manipulation with altered zinc availability to assess zinc-dependence of observed phenotypes

What approaches can resolve contradictory data on SLC30A5 expression across different studies?

Resolving contradictory findings on SLC30A5 expression requires systematic approaches:

• Meta-analysis of existing data:

  • Comprehensively review expression data across multiple platforms (RNA-seq, microarray, proteomics)

  • Account for differences in tissue sources, processing methods, and detection techniques

  • Use statistical approaches to identify consistent patterns despite methodological variations

• Technical validation:

  • Employ multiple detection methods (qPCR, Western blot, IHC) within the same study

  • Use different antibodies targeting distinct epitopes of SLC30A5

  • Include appropriate positive and negative controls with known SLC30A5 expression levels

• Biological context considerations:

  • Account for zinc status of experimental systems, as zinc levels can regulate expression of zinc transporters

  • Consider dynamic expression changes in response to stimuli (e.g., LPS can transiently downregulate SLC30A5 in human monocyte-derived macrophages)

  • Evaluate cell type-specific expression patterns using single-cell sequencing approaches

• Standardized reporting:

  • Precisely document experimental conditions, particularly zinc concentrations in media and serum

  • Report antibody validation data, including verification of specificity

  • Clearly describe quantification methods and normalization approaches

How can I assess the relationship between SLC30A5 expression and immune cell function?

The relationship between SLC30A5 and immune function represents an emerging research area that can be investigated through several approaches:

• Expression analysis in immune populations:

  • Analyze SLC30A5 expression across immune cell subsets using flow cytometry and cell sorting followed by qPCR or Western blotting

  • Monitor expression changes during immune cell activation, differentiation, or in response to pathogen-associated molecular patterns like LPS

• Correlation with immune parameters:

  • Use tools like TIMER 2.0 database (http://timer.comp-genomics.org) to assess correlations between SLC30A5 expression and immune cell infiltration in tumors

  • Analyze relationships between SLC30A5 expression and immune checkpoint molecules, which may influence anti-tumor immunity

• Functional studies:

  • Generate immune cells with SLC30A5 knockdown or overexpression

  • Assess standard immune functions such as cytokine production, phagocytosis, antigen presentation, and migration

  • Evaluate zinc-dependent immune pathways specifically, as SLC30A family members regulate zinc availability for antimicrobial responses

• Co-localization with immune compartments:

  • Determine if SLC30A5 localizes to specialized immune compartments such as phagosomes

  • Assess whether SLC30A5 contributes to zinc trafficking during immune responses, similar to the antimicrobial zinc toxicity mechanism demonstrated for SLC30A1 in macrophages

What are the most effective approaches to study SLC30A5 as a therapeutic target in cancer?

To investigate SLC30A5 as a therapeutic target in cancer, consider these methodological approaches:

• Target validation studies:

  • Perform comprehensive knockdown/knockout experiments in multiple cancer cell lines

  • Evaluate effects on key cancer hallmarks: proliferation, apoptosis resistance, migration, invasion, and metabolism

  • Use xenograft models to confirm in vitro findings regarding tumor growth and metastasis

• Molecular targeting strategies:

  • Develop monoclonal antibodies targeting extracellular domains of SLC30A5

  • Design small molecule inhibitors of zinc transport function

  • Explore siRNA/shRNA therapeutic delivery systems for gene silencing in tumors

• Combination therapy investigations:

  • Test SLC30A5 inhibition in combination with standard chemotherapeutics

  • Evaluate synergy with immune checkpoint inhibitors, particularly given the correlations between SLC30A5 expression and immune cell infiltration

  • Examine combinations with zinc chelators or zinc supplementation strategies

• Biomarker development:

  • Develop immunohistochemical protocols for SLC30A5 assessment in patient biopsies

  • Create standardized scoring systems for SLC30A5 expression

  • Correlate expression with treatment response and patient outcomes in retrospective and prospective studies

• Resistance mechanisms:

  • Investigate compensatory upregulation of other zinc transporters following SLC30A5 inhibition

  • Identify bypass pathways that may confer resistance to SLC30A5-targeted therapies

  • Develop strategies to overcome potential resistance mechanisms