SLC39A4 Antibody, FITC conjugated

What is SLC39A4 and why is it important for research?

SLC39A4, also known as ZIP4 or Zrt- and Irt-like protein 4, functions as a critical zinc transporter that regulates cellular zinc uptake . This protein plays an essential role in maintaining zinc homeostasis, a process fundamental to numerous physiological functions. Zinc is an essential micronutrient involved in protein structure, enzymatic activity, gene expression, and cell signaling pathways . SLC39A4's importance is highlighted by its association with various pathological conditions, including cancer and metabolic disorders, making it a significant target for biomedical research . The protein is particularly interesting due to evolutionary adaptations, as evidenced by extreme allele frequency differences between West African and Eurasian populations for a leucine-to-valine substitution (Leu372Val) .

What are the key specifications of commercially available SLC39A4 antibodies?

SLC39A4 antibodies are available in various formats, with FITC-conjugated rabbit polyclonal antibodies being particularly useful for immunofluorescence applications . These antibodies typically recognize specific epitopes of the SLC39A4 protein and are validated for use in multiple species, most commonly human, mouse, and rat models . The antibodies are generally supplied in liquid form with stabilizing buffers containing glycerol and preservatives like Proclin 300 . Storage recommendations typically specify -20°C or -80°C to maintain optimal reactivity . The immunogens used for antibody production often consist of recombinant fusion proteins containing sequences corresponding to specific amino acid regions of human SLC39A4, such as amino acids 23-327 (NP_570901.2) .

What applications are SLC39A4 antibodies validated for?

SLC39A4 antibodies have been validated for multiple research applications, providing versatility in experimental approaches:

For optimal results, each laboratory should determine the ideal dilution for their specific experimental conditions .

How should I optimize staining protocols for FITC-conjugated SLC39A4 antibodies?

When optimizing staining protocols with FITC-conjugated SLC39A4 antibodies, several factors require careful consideration. First, establish appropriate negative controls using isotype-matched control antibodies to assess non-specific binding . For fixed tissue sections or cells, optimize fixation conditions to preserve epitope accessibility while maintaining cellular architecture—typically 4% paraformaldehyde works well for FITC-conjugated antibodies .

Titrate antibody concentrations to determine optimal signal-to-noise ratio, starting with manufacturer recommendations (typically 1:50-1:100 for immunofluorescence) . Include antigen retrieval steps for formalin-fixed paraffin-embedded (FFPE) tissues, usually citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Minimize photobleaching by limiting exposure to light during incubation and washing steps, and consider mounting media containing anti-fade agents . If cross-reactivity is observed, implement additional blocking steps using sera from the same species as the secondary antibody or add protein blockers like BSA or casein.

What controls should be included when using SLC39A4 antibodies in cancer research?

When investigating SLC39A4 in cancer contexts, comprehensive control strategies are essential. Include positive control tissues known to express SLC39A4, such as intestinal epithelium or pancreatic β-cells for validating antibody performance . Incorporate negative control tissues that lack SLC39A4 expression to confirm specificity. For cellular studies, include cell lines with verified high SLC39A4 expression (e.g., Hepa 1-6 mouse hepatoma cell line) and those with low expression as comparative controls .

Implement siRNA knockdown or CRISPR knockout of SLC39A4 to generate negative control samples that demonstrate antibody specificity . Use matched normal-tumor tissue pairs to assess differential expression in cancer progression. When evaluating therapeutic responses, include treatment time courses and dose-response analyses to comprehensively characterize SLC39A4 dynamics . In chemoresistance studies, compare SLC39A4 expression between resistant and sensitive cell populations, as SLC39A4 has been implicated in resistance to cisplatin in esophageal squamous cell carcinoma .

How can I troubleshoot weak or absent signals when using SLC39A4 antibodies?

When encountering weak or absent signals with SLC39A4 antibodies, systematic troubleshooting is required. First, verify antibody viability by checking storage conditions and expiration date—improper storage or freeze-thaw cycles can compromise activity . Optimize antigen retrieval methods, considering that SLC39A4 epitopes may be sensitive to particular fixation methods—test both heat-induced (citrate or EDTA buffers) and enzymatic retrieval approaches .

Increase antibody concentration gradually, adjusting from standard dilutions (e.g., 1:1000 for WB) to more concentrated applications (e.g., 1:500 or 1:250) . Extend primary antibody incubation time, potentially incorporating overnight incubation at 4°C rather than shorter incubations at room temperature. Evaluate sample preparation techniques, as some lysis buffers may affect the SLC39A4 epitope—consider testing alternate extraction protocols with different detergents . If using fluorescent detection methods, adjust microscope settings to optimize signal detection, and use positive control tissues with known high SLC39A4 expression to benchmark proper technique .

How can SLC39A4 antibodies be utilized to investigate zinc transport mechanisms?

To investigate zinc transport mechanisms using SLC39A4 antibodies, researchers can implement several sophisticated approaches. Dual immunofluorescence labeling can be performed using FITC-conjugated SLC39A4 antibodies alongside intracellular zinc indicators (like FluoZin-3) to correlate transporter localization with zinc concentration gradients . Live-cell imaging can track SLC39A4 trafficking in response to extracellular zinc fluctuations, as SLC39A4 is known to relocalize between the plasma membrane and endosomal compartments depending on zinc availability .

Super-resolution microscopy techniques (STORM, PALM, or STED) combined with FITC-conjugated SLC39A4 antibodies can reveal nanoscale organization of zinc transporters at the cell membrane . Co-immunoprecipitation studies using SLC39A4 antibodies can identify protein interaction partners that regulate transporter function or trafficking. Pulse-chase experiments with surface biotinylation can determine SLC39A4 turnover rates and how these are affected by zinc status. Additionally, patch-clamp electrophysiology combined with immunolocalization can correlate transporter distribution with functional zinc current measurements .

What role does SLC39A4 play in cancer progression and how can antibodies help investigate this?

SLC39A4 has emerged as a significant player in cancer progression, particularly in esophageal squamous cell carcinoma (ESCC) where it demonstrates oncogenic properties . Antibody-based research approaches can elucidate several aspects of SLC39A4's role in cancer. Tissue microarray analysis using SLC39A4 antibodies can assess expression patterns across tumor stages, revealing correlations with clinical outcomes as demonstrated by studies showing that high SLC39A4 expression correlates with advanced clinical stage (p=0.023) and T categories (p=0.035) in ESCC patients .

Multiplexed immunofluorescence combining SLC39A4 antibodies with markers of EMT (epithelial-mesenchymal transition) such as E-cadherin, N-cadherin, Vimentin, and Snail can reveal mechanistic connections between zinc transport and metastatic potential . Chemoresistance mechanisms can be investigated by correlating SLC39A4 expression with treatment response, as studies indicate SLC39A4 strengthens resistance of ESCC cells to cisplatin in vitro . Xenograft models with SLC39A4-manipulated cancer cells can be analyzed using immunohistochemistry to track zinc transporter expression during tumor development in vivo. Proximity ligation assays utilizing SLC39A4 antibodies can identify novel protein interactions specific to cancer cells that may represent therapeutic targets .

How can genetic variation in SLC39A4 impact antibody selection and experimental design?

Genetic variations in SLC39A4, such as the leucine-to-valine substitution (Leu372Val) with extreme population differences between West Africans and Eurasians, necessitate careful antibody selection and experimental design considerations . When working with populations of diverse genetic backgrounds, researchers should verify that selected antibodies recognize epitopes conserved across known variants to avoid false negatives in polymorphic regions .

For studies involving population genetics, western blot validation of antibody recognition across samples with different SLC39A4 genotypes is recommended. Consider epitope mapping to determine if antibodies target regions with known polymorphisms, particularly when working with specific isoforms . When investigating functional consequences of genetic variants, pair antibody-based expression studies with genetic sequencing to correlate genotype with protein expression patterns .

In evolutionary biology research, combine population-specific antibody validation with analyses of selection signatures to understand functional adaptations in zinc metabolism across human populations. For clinical applications, stratify samples by genotype before analyzing SLC39A4 expression to account for variant-specific differences in antibody affinity or protein function . Additionally, when investigating disease associations, consider how genetic variants might affect antibody recognition or protein localization, potentially necessitating complementary approaches like mRNA quantification .

How can I combine SLC39A4 antibody-based approaches with genomic and transcriptomic analyses?

Integrating SLC39A4 antibody-based approaches with genomic and transcriptomic analyses creates a comprehensive multi-omics framework for investigating zinc transport biology. ChIP-seq (Chromatin Immunoprecipitation Sequencing) using antibodies against transcription factors regulating SLC39A4 can be correlated with RNA-seq data to identify regulatory networks controlling zinc transporter expression . Single-cell approaches combining immunofluorescence detection of SLC39A4 protein with single-cell RNA-seq can reveal cell-type-specific expression patterns and heterogeneity within tissues.

For population studies, correlate genotypic data from genome-wide association studies (GWAS) with SLC39A4 protein expression measured by immunohistochemistry to identify expression quantitative trait loci (eQTLs) . Spatial transcriptomics paired with immunofluorescence can map the relationship between SLC39A4 transcript and protein distribution within complex tissue architectures. In disease studies, integrate proteogenomic approaches by correlating SLC39A4 protein levels with somatic mutation or copy number variation data in cancer samples . Additionally, CRISPR screens targeting zinc homeostasis genes can be evaluated using SLC39A4 antibodies to assess downstream effects on protein expression and localization .

What are the best approaches for quantifying SLC39A4 expression in different experimental models?

Quantification of SLC39A4 expression across experimental models requires tailored methodological approaches. For cell culture systems, western blotting with validated SLC39A4 antibodies followed by densitometric analysis provides reliable relative quantification, as demonstrated with Hepa 1-6 mouse hepatoma and D3 mouse embryonic stem cell lines . Flow cytometry using FITC-conjugated SLC39A4 antibodies enables single-cell quantification and can identify subpopulations with differential expression levels .

In tissue sections, digital image analysis of immunohistochemistry can provide semi-quantitative data on expression levels and subcellular localization patterns. Recommended approaches include tissue microarrays for high-throughput analysis across multiple samples . For absolute quantification, ELISA-based methods using calibrated standard curves can determine SLC39A4 concentration in protein lysates . In complex tissues, multiplexed immunofluorescence combined with confocal microscopy and computational image analysis allows quantification of SLC39A4 in specific cell types within heterogeneous populations.

When comparing expression across different experimental models, normalization strategies are essential—normalize to total protein load for western blots, use housekeeping genes like GAPDH as loading controls, and include reference cell lines with stable SLC39A4 expression as inter-experimental calibrators .

How can SLC39A4 antibodies contribute to studying chemotherapy resistance mechanisms?

SLC39A4 antibodies provide valuable tools for investigating chemotherapy resistance mechanisms, particularly in cancers where zinc homeostasis affects treatment response. Immunoblotting with SLC39A4 antibodies can track changes in expression levels before and after chemotherapy exposure, revealing adaptive responses to treatment stress . Confocal microscopy using fluorescently-labeled antibodies can monitor subcellular redistribution of SLC39A4 during development of resistance, as transporter localization may change to alter intracellular zinc levels.

Flow cytometry combining SLC39A4 antibodies with apoptotic markers can correlate expression levels with cell death responses in heterogeneous tumor populations . In patient-derived xenograft models, immunohistochemistry can evaluate if SLC39A4 expression patterns predict treatment response, potentially identifying predictive biomarkers. Research has demonstrated that silencing SLC39A4 significantly increases the apoptosis rate of esophageal squamous cell carcinoma cells exposed to cisplatin, while overexpression inhibits cisplatin-mediated apoptosis .

For mechanistic studies, co-immunoprecipitation with SLC39A4 antibodies can identify binding partners involved in drug resistance pathways. Combining zinc chelators like TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine) with SLC39A4 expression analysis can determine if zinc availability directly mediates resistance mechanisms . Time-course studies measuring SLC39A4 expression during stepwise development of resistance can identify when transporter upregulation occurs in relation to phenotypic resistance .

How might SLC39A4 antibodies contribute to therapeutic development targeting zinc transport?

SLC39A4 antibodies hold significant potential for therapeutic development targeting zinc transport mechanisms. Antibody-drug conjugates (ADCs) could be developed using SLC39A4 antibodies to deliver cytotoxic agents specifically to cells overexpressing this transporter, such as certain cancer cells . Screening assays employing fluorescence polarization with labeled SLC39A4 antibodies can identify small molecule inhibitors that modulate zinc transporter function or expression.

Neutralizing antibodies against extracellular domains of SLC39A4 might be developed to block zinc uptake in pathological conditions where excessive zinc contributes to disease progression, particularly in cancers where SLC39A4 overexpression correlates with poor prognosis . Intrabodies (intracellular antibodies) targeting SLC39A4 could be engineered to alter its trafficking between plasma membrane and endosomal compartments, thereby modulating zinc uptake capacity .

For diagnostic applications, imaging agents based on SLC39A4 antibodies could help identify tumors with altered zinc metabolism. Therapeutic strategies combining zinc chelators with SLC39A4 inhibition might enhance chemosensitivity in resistant tumors, as research has shown knockdown of SLC39A4 increases cisplatin sensitivity . Additionally, antibody-based tools could help validate SLC39A4 as a therapeutic target by mapping its expression across healthy and diseased tissues to predict potential side effects of targeted interventions .

What challenges exist in developing new SLC39A4 antibodies for specialized research applications?

Developing specialized SLC39A4 antibodies presents several technical challenges. Creating antibodies targeting specific SLC39A4 conformations associated with active zinc transport versus inactive states requires sophisticated structural information and screening methods. Producing antibodies that distinguish between SLC39A4 and other zinc transporters in the same family (ZIP family) demands careful epitope selection to avoid cross-reactivity, as these proteins share significant sequence homology .

Generating antibodies against post-translationally modified SLC39A4 (phosphorylated, ubiquitinated, or glycosylated forms) requires purification or synthesis of appropriately modified immunogens. For developing antibodies to specific genetic variants, such as the Leu372Val polymorphism with population differences, researchers must create variant-specific immunogens and implement rigorous validation protocols .

Creating non-perturbing antibodies for live-cell imaging applications presents another challenge, as these must bind SLC39A4 without altering its function or localization. Furthermore, developing antibodies compatible with super-resolution microscopy techniques requires specific fluorophore conjugation strategies that maintain antibody affinity while providing optimal photophysical properties . The membrane-embedded nature of SLC39A4 with multiple transmembrane domains limits accessible epitopes, making antibody development against specific functional regions particularly challenging .

How can SLC39A4 antibodies contribute to understanding population differences in zinc metabolism?

SLC39A4 antibodies offer valuable tools for investigating population differences in zinc metabolism, particularly given the documented genetic variation across human populations . Immunohistochemistry studies using SLC39A4 antibodies across tissue samples from diverse populations can map expression patterns correlated with genotypic data, potentially revealing functional adaptations in zinc transport systems. Western blot analysis comparing protein expression levels between population groups with different allele frequencies for the Leu372Val variant can determine if genetic polymorphisms translate to altered protein abundance .

Functional assays combining zinc transport measurements with SLC39A4 localization studies can reveal whether population-specific variants alter transporter efficiency or regulation. Antibodies recognizing specific SLC39A4 variants could be developed to directly compare the expression and localization of different isoforms within heterozygous individuals. In cellular models, FITC-conjugated SLC39A4 antibodies can track real-time responses to zinc challenges in cells engineered to express population-specific variants, potentially revealing differences in trafficking dynamics .

Correlative studies between SLC39A4 expression patterns and population-specific disease susceptibilities might uncover links between zinc metabolism adaptation and health outcomes. Additionally, antibody-based proteomics approaches could identify population differences in SLC39A4 interaction networks that might explain adaptive advantages in different nutritional environments throughout human evolutionary history .