slc30a9 Antibody

What is SLC30A9 and why is it important in biological research?

SLC30A9 (Solute Carrier Family 30 Member 9) is an evolutionarily conserved housekeeping gene that encodes a zinc transporter protein, also known as ZnT-9. It plays critical roles intimately linked with cellular replication, DNA synthesis, and transcriptional regulation. The protein functions as a proton-coupled zinc antiporter located in mitochondria and is essential for zinc homeostasis and mobilization within cells . Due to its fundamental role in zinc metabolism and cellular processes, SLC30A9 has become an important target for research in areas including cellular biology, neuroscience, and disease pathophysiology studies where zinc dysregulation is implicated.

What are the most common alternative names for SLC30A9 in the scientific literature?

When searching for literature on SLC30A9, researchers should be aware of its multiple aliases to ensure comprehensive results. Protein aliases include: expressed in human embryonic lung, GAC63, GRIP1-associated coactivator 1, GRIP1-associated coactivator 63, GRIP1-dependent nuclear receptor coactivator, HuEL, Human embryonic lung protein, Proton-coupled zinc antiporter SLC30A9, mitochondrial, Zinc transporter 9, and ZnT-9 . Gene aliases include: 2310024J23Rik, AL024256, C4orf1, GAC63, HUEL, and ZNT9 . Using these alternative names in literature searches will yield more comprehensive results when reviewing existing research on this protein.

What is the molecular weight and structure of the human SLC30A9 protein?

The human SLC30A9 protein has a molecular weight of approximately 63.51 kDa . Structurally, SLC30A9 shares extensive sequence homology with the bacterial Zn²⁺/H⁺ exchanger YiiP , suggesting similar functional mechanisms. The protein contains multiple transmembrane domains characteristic of membrane transporters. The gene is encoded on chromosome 4 in humans, and the protein's structure facilitates its function as a zinc transporter, particularly in mitochondria where it appears to be involved in zinc export mechanisms . This structural information is important when selecting antibodies and designing experiments targeting specific domains of the protein.

What types of SLC30A9 antibodies are available for research applications?

Both polyclonal and monoclonal antibodies against SLC30A9 are available for research. Polyclonal antibodies include those derived from rabbit that detect endogenous levels of total SLC30A9/ZnT-9 protein . These antibodies are typically generated against synthesized peptides from various regions of human SLC30A9, including internal regions. Monoclonal antibodies such as PCRP-SLC30A9-1C3 (mouse IgG2b isotype) are also available, generated against full-length recombinant human SLC30A9 protein . When selecting an antibody, researchers should consider factors such as the host species, clonality, epitope location, and validated applications to ensure optimal results for their specific experimental needs.

How should researchers validate the specificity of an SLC30A9 antibody before experimental use?

Proper validation of SLC30A9 antibodies should include multiple approaches. The gold standard is comparing antibody reactivity between wild-type and SLC30A9 knockout cells, as demonstrated in studies using SLC30A9⁻/⁻ HeLa cells . Additional validation methods include: (1) Western blotting to confirm detection of a band at the expected molecular weight (~63 kDa); (2) Peptide competition assays where pre-incubation with the immunizing peptide should abolish specific signals; (3) RNA interference to correlate protein detection with gene expression knockdown; (4) Testing for cross-reactivity with other zinc transporters, particularly other SLC30 family members; and (5) Immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein. Thorough validation ensures experimental observations are truly related to SLC30A9 and not due to non-specific binding.

Which SLC30A9 antibody applications are most reliably validated in the literature?

Based on the available information, Western blotting (WB) appears to be the most consistently validated application for SLC30A9 antibodies . Both polyclonal antibodies have demonstrated efficacy in WB at dilutions ranging from 1:500 to 1:2000 . Additionally, ELISA applications have been validated for several antibodies at dilutions up to 1:20000 . For cellular localization studies, immunocytochemistry (ICC) and immunofluorescence (IF) applications have been validated for some antibodies . The monoclonal antibody PCRP-SLC30A9-1C3 is specifically recommended for immunoprecipitation and microarray applications . For advanced applications such as chromatin immunoprecipitation or tissue staining, additional validation by individual researchers is strongly recommended before proceeding with large-scale experiments.

How should researchers design experiments to study SLC30A9 subcellular localization?

To effectively study SLC30A9 subcellular localization, researchers should implement a multi-method approach. Begin with immunofluorescence microscopy using validated antibodies against SLC30A9 combined with established organelle markers, particularly mitochondrial markers since SLC30A9 has been shown to localize to mitochondria . Counterstain with DAPI for nuclear visualization. For higher resolution, super-resolution microscopy techniques like STED or STORM can reveal detailed localization patterns. Complement optical techniques with subcellular fractionation followed by Western blotting to biochemically confirm the presence of SLC30A9 in specific cellular compartments. For definitive localization studies, electron microscopy with immunogold labeling provides nanometer-scale precision. Additionally, expression of fluorescent protein-tagged SLC30A9 (ensuring the tag doesn't interfere with localization) can enable live-cell imaging to study dynamic localization patterns in response to stimuli such as zinc level fluctuations.

What controls are essential when using SLC30A9 antibodies for Western blotting experiments?

Several controls are essential when using SLC30A9 antibodies for Western blotting: (1) Positive control: Lysates from cells known to express SLC30A9 (most human cell lines express the protein); (2) Negative control: Lysates from SLC30A9 knockout cells or cells where SLC30A9 has been knocked down using siRNA/shRNA ; (3) Loading control: Probing for housekeeping proteins (β-actin, GAPDH) to ensure equal loading across samples; (4) Molecular weight marker: To confirm the detected band matches the expected molecular weight of ~63 kDa for human SLC30A9 ; (5) Peptide competition control: Pre-incubating the antibody with the immunizing peptide should eliminate specific bands; (6) Secondary antibody-only control: To identify non-specific binding of the secondary antibody; and (7) Recombinant protein: Purified SLC30A9 protein can serve as a reference for band size and antibody reactivity. These controls collectively ensure the specificity and reliability of Western blotting results.

What are the optimal conditions for immunoprecipitation of SLC30A9?

For successful immunoprecipitation (IP) of SLC30A9, several parameters should be optimized. Cell lysis should be performed using a non-denaturing buffer (e.g., RIPA or NP-40 based) supplemented with protease inhibitors and potentially zinc chelators depending on experimental goals. The monoclonal antibody PCRP-SLC30A9-1C3 is specifically recommended for IP applications . For coupling the antibody, Protein G-conjugated beads are suitable for mouse monoclonal antibodies, while Protein A works well for rabbit polyclonals. Pre-clearing lysates with naked beads reduces non-specific binding. Antibody-to-lysate ratios should be optimized (starting with ~2-5 μg antibody per 500 μg protein), with incubation at 4°C overnight with gentle rotation. Multiple gentle washes with decreasing salt concentrations help minimize background while preserving specific interactions. Elution can be performed using low pH glycine buffer, SDS sample buffer, or specific peptide competition depending on downstream applications. IP efficiency should be verified by Western blotting, comparing input, unbound, and immunoprecipitated fractions.

How can SLC30A9 antibodies be used to study zinc transport mechanisms in mitochondria?

SLC30A9 antibodies can be instrumental in elucidating mitochondrial zinc transport mechanisms through several sophisticated approaches. Researchers can combine immunofluorescence with zinc-specific fluorescent probes (such as FluoZin-3) to correlate SLC30A9 localization with zinc levels in live cells. Proximity ligation assays (PLA) using SLC30A9 antibodies paired with antibodies against other mitochondrial proteins can identify potential interaction partners in the zinc transport machinery. For functional studies, researchers can perform zinc flux assays in isolated mitochondria from wild-type versus SLC30A9-depleted cells, using the antibody to confirm knockout/knockdown efficiency . Advanced techniques like FRET-based zinc sensors targeted to mitochondria can monitor real-time zinc movement in relation to SLC30A9 expression or manipulation. Additionally, immunoprecipitation of SLC30A9 followed by mass spectrometry can identify associated proteins that may be part of a larger zinc transport complex. These multifaceted approaches collectively provide mechanistic insights into how SLC30A9 functions as a Zn²⁺/H⁺ exchanger in mitochondrial membranes.

What strategies should be employed when antibody signals for endogenous SLC30A9 are weak?

When facing weak antibody signals for endogenous SLC30A9, researchers should implement a systematic troubleshooting approach. First, consider signal amplification techniques such as using biotin-streptavidin systems, tyramide signal amplification, or highly sensitive ECL substrates for Western blotting. Sample preparation optimization is crucial—evaluate different lysis buffers that might better preserve the protein's native state and epitopes, and consider zinc supplementation or depletion during cell culture, as this might affect SLC30A9 expression levels. Test multiple antibodies targeting different epitopes of SLC30A9, as protein modifications or conformational changes may mask certain epitopes. For immunofluorescence, try antigen retrieval methods, longer primary antibody incubation times (overnight at 4°C), and confocal microscopy for improved signal detection. If the issue persists, consider enriching the protein through immunoprecipitation before detection or implementing more sensitive detection methods like digital PCR to correlate protein levels with mRNA expression. As a last resort, creating a cell line overexpressing tagged SLC30A9 can provide a positive control system to optimize detection conditions.

How can researchers differentiate between SLC30A9 and other zinc transporters in their experiments?

Differentiating between SLC30A9 and other zinc transporters requires multiple parallel approaches to ensure specificity. Antibody selection is critical—researchers should use antibodies raised against unique regions of SLC30A9 with minimal sequence homology to other SLC30 family members. Verification through siRNA-mediated selective knockdown of SLC30A9 followed by Western blotting helps confirm antibody specificity. For expression studies, designing PCR primers that span unique regions of the SLC30A9 transcript ensures selective amplification. When studying protein function, researchers should combine genetic approaches (CRISPR-Cas9 knockout of SLC30A9) with pharmacological inhibitors that may differentiate between transporter subtypes. Co-immunoprecipitation experiments followed by mass spectrometry can identify unique interaction partners of SLC30A9 compared to other zinc transporters. For localization studies, the distinct mitochondrial localization of SLC30A9 contrasts with other family members that localize to different cellular compartments, providing another differentiation parameter. Finally, functional zinc transport assays with organelle-specific zinc probes can help distinguish the specific contribution of SLC30A9 to zinc homeostasis in different cellular compartments.

How should researchers address cross-reactivity issues with SLC30A9 antibodies?

When encountering cross-reactivity with SLC30A9 antibodies, researchers should implement a systematic verification process. First, perform an epitope analysis by comparing the immunizing peptide sequence against protein databases to identify potentially similar sequences in other proteins. Test the antibody on samples from SLC30A9 knockout models—a clean antibody should show no signal in these samples . To confirm specificity biochemically, perform peptide competition assays using both the specific immunizing peptide and peptides from potentially cross-reactive proteins. Western blotting across multiple cell lines with different expression profiles of related proteins can help identify patterns of cross-reactivity. For applications like immunohistochemistry, include tissue from knockout animals or use RNA scope as a complementary approach to verify protein localization patterns. If cross-reactivity persists, consider using multiple antibodies targeting different epitopes of SLC30A9 and look for overlapping signals as confirmation. As a last resort, immunodepletion of the antibody using recombinant proteins from suspected cross-reactive species can help enrich for SLC30A9-specific antibodies in the preparation.

What are the critical factors affecting SLC30A9 antibody performance in different experimental contexts?

Several critical factors influence SLC30A9 antibody performance across experimental platforms. Sample preparation methods significantly impact epitope accessibility—native protein conformation is better preserved in non-denaturing techniques (IP, IF) while denatured proteins expose different epitopes (Western blotting). Fixation methods for immunocytochemistry/immunohistochemistry can dramatically affect antibody binding, with paraformaldehyde potentially masking certain epitopes that might be accessible with methanol fixation. Buffer composition is crucial—pH, salt concentration, detergents, and the presence of zinc ions can all influence antibody-epitope interactions. For polyclonal antibodies, lot-to-lot variation may be significant , requiring validation of each new lot. The cellular activation state may affect SLC30A9 post-translational modifications, potentially masking antibody epitopes under certain conditions. Importantly, cross-species reactivity varies between antibodies—while some react with human, mouse, and rat SLC30A9 , others may be species-specific . Temperature and incubation time need optimization for each application, with longer, colder incubations (4°C overnight) often improving signal-to-noise ratios compared to shorter incubations at room temperature.

How can inconsistencies in SLC30A9 antibody results between different experimental techniques be reconciled?

Inconsistencies between techniques using SLC30A9 antibodies often stem from fundamental differences in how each method presents the antigen. To reconcile such discrepancies, researchers should first understand that different techniques expose different epitopes—Western blotting detects denatured linear epitopes, while immunofluorescence targets native conformational epitopes. Test multiple antibodies targeting different regions of SLC30A9 across techniques to build a comprehensive picture. When Western blotting shows a band but immunofluorescence shows no signal, consider that the epitope might be masked in the native state or that fixation methods are affecting accessibility. Conversely, if immunofluorescence works but Western blotting fails, the epitope might be dependent on tertiary structure. Create a positive control system with tagged SLC30A9 overexpression to validate detection methods across platforms. For functional studies, combine antibody-based detection with complementary techniques like RNA expression analysis or activity assays to correlate protein detection with functional outcomes. When publishing results, researchers should explicitly describe all validation steps performed and acknowledge any limitations in antibody performance across different applications, providing a nuanced interpretation of the collective data rather than relying on a single technique.

How can SLC30A9 antibodies be used to investigate the protein's role in disease models?

SLC30A9 antibodies serve as valuable tools for investigating this protein's role in disease models through multiple experimental approaches. In neurodegenerative disease models where zinc dyshomeostasis is implicated, immunohistochemistry with SLC30A9 antibodies can map expression changes in affected brain regions. For cancer research, tissue microarray analysis using validated antibodies can identify correlation between SLC30A9 expression levels and disease progression or treatment response. In genetic disorders, combining SLC30A9 immunodetection with functional zinc imaging can reveal mechanisms linking gene mutations to cellular pathophysiology. For inflammatory conditions, tracking SLC30A9 subcellular redistribution during inflammatory stimulation may reveal stress-response mechanisms. When studying animal models, antibodies validated for cross-species reactivity allow translation between preclinical models and human samples . For therapeutic development, SLC30A9 antibodies can monitor protein modulation in response to drug candidates targeting zinc homeostasis. When interpreting disease-related findings, researchers should correlate protein expression data with functional outcomes using zinc sensors or mitochondrial function assays to establish causative relationships rather than mere associations between SLC30A9 alterations and disease phenotypes.

What approaches should be used to analyze SLC30A9 protein interactions with other cellular components?

Analyzing SLC30A9 protein interactions requires multiple complementary techniques. Co-immunoprecipitation using antibodies like PCRP-SLC30A9-1C3 (recommended for IP) followed by mass spectrometry can identify novel interaction partners. Proximity ligation assays provide in situ visualization of protein-protein interactions with spatial resolution, while FRET/BRET approaches can detect interactions in live cells. For mapping interaction domains, researchers can use truncated constructs combined with IP to identify specific binding regions. Yeast two-hybrid or mammalian two-hybrid systems offer alternative approaches for detecting binary interactions. To understand functional significance, researchers should correlate interaction data with functional outcomes using zinc transport assays or mitochondrial function tests in cells where specific interactions are disrupted. Crosslinking followed by IP (CLIP) can stabilize transient interactions before analysis. Importantly, researchers should validate interactions under physiologically relevant conditions, including manipulating zinc concentrations to determine if interactions are zinc-dependent. When studying mitochondrial interactions, subcellular fractionation should precede IP to enrich for mitochondrial proteins and reduce cytoplasmic contaminants. Results from multiple methods should be integrated to build confidence in the biological relevance of identified interactions.

How should researchers interpret differences in SLC30A9 expression across different tissues and cell types?

Interpreting SLC30A9 expression variations across tissues and cell types requires contextual analysis beyond mere quantification. Researchers should first establish a quantitative baseline using techniques like Western blotting with carefully validated antibodies , ideally calibrated against recombinant standards. This should be complemented with mRNA quantification to distinguish transcriptional from post-transcriptional regulation. When comparing tissues, consider their metabolic activity and zinc requirements—high SLC30A9 expression may correlate with tissues having elevated mitochondrial activity or zinc flux. Cell-type specific expression should be evaluated using immunohistochemistry or single-cell approaches to avoid averaging effects in heterogeneous tissues. Developmental timing is crucial, as SLC30A9 expression may change throughout organ development and aging. Functional correlation is essential—higher expression doesn't necessarily mean higher activity, so zinc transport assays should complement expression data. For disease-related studies, compare expression changes to established disease markers and clinical outcomes. When publishing such comparisons, always include detailed methodological validation and use consistent techniques across samples to ensure differences reflect biological variation rather than technical artifacts. This multi-level analysis provides more meaningful interpretation than simple statements about relative expression levels.

What emerging techniques might enhance the utility of SLC30A9 antibodies in zinc transport research?

Several cutting-edge techniques are poised to revolutionize SLC30A9 antibody applications in zinc transport research. Lattice light-sheet microscopy combined with specific antibodies could enable real-time tracking of SLC30A9 dynamics in live cells with minimal phototoxicity. CRISPR epitope tagging of endogenous SLC30A9 followed by anti-tag antibodies offers a strategy to circumvent specificity issues with direct antibodies. Expanding to spatial transcriptomics and proteomics, researchers could correlate SLC30A9 protein localization with gene expression profiles at the single-cell level. Nanobodies derived from conventional SLC30A9 antibodies might provide superior penetration for intracellular tracking. Cryo-electron tomography with immunogold-labeled antibodies could reveal SLC30A9's native structure in the mitochondrial membrane at near-atomic resolution. For functional studies, optogenetic control of SLC30A9 coupled with antibody-based detection methods could allow precise temporal correlation between protein activation and zinc transport. Mass cytometry (CyTOF) using metal-conjugated SLC30A9 antibodies would enable high-dimensional analysis of zinc transport networks across heterogeneous cell populations. These advanced techniques, when combined with established antibody applications, will provide unprecedented insights into the spatial, temporal, and functional aspects of SLC30A9-mediated zinc transport in health and disease contexts.

How can SLC30A9 antibodies contribute to understanding the protein's role in mitochondrial function and disease?

SLC30A9 antibodies are becoming essential tools for unraveling this protein's critical role in mitochondrial pathophysiology. By combining super-resolution microscopy with specific antibodies, researchers can map SLC30A9's precise localization within mitochondrial subcompartments. In neurodegenerative diseases with mitochondrial dysfunction, SLC30A9 immunodetection in patient-derived samples can reveal disease-specific alterations in zinc transport machinery. For dynamic studies, correlative live-cell imaging with post-fixation immunolabeling enables tracking of mitochondrial morphological changes in relation to SLC30A9 distribution. In metabolic disorders, antibody-based tissue microarrays can establish relationships between SLC30A9 expression patterns and metabolic parameters across patient cohorts. Functionally, researchers can use antibodies to measure SLC30A9 expression or modification in response to mitochondrial stressors, correlating protein changes with functional outcomes measured by respirometry or membrane potential assays. The unique position of SLC30A9 as a mitochondrial zinc transporter makes antibodies against it valuable for studying zinc's largely unexplored role in mitochondrial biology—from electron transport chain function to mitochondrial dynamics and quality control mechanisms that may be disrupted in various disease states.

What standardization practices should the research community adopt for SLC30A9 antibody-based research?

To advance SLC30A9 research, the scientific community should implement comprehensive standardization practices. A centralized antibody validation registry specifically for SLC30A9 antibodies would allow researchers to share validation data across applications and experimental conditions. Standardized reporting guidelines should include mandatory disclosure of antibody catalog numbers, lot numbers, dilutions, incubation conditions, and all optimization steps. Reference materials including recombinant SLC30A9 protein standards and SLC30A9 knockout cell lysates should be developed and made widely available as benchmarking tools. Multisite reproducibility initiatives focusing specifically on SLC30A9 detection methods would identify robust protocols that produce consistent results across laboratories. Digital image acquisition and analysis parameters should be standardized and fully reported to enable quantitative comparisons between studies. Round-robin testing of commercially available antibodies would provide objective performance rankings across applications. For immunohistochemistry, standardized scoring systems for SLC30A9 expression should be developed and validated. Journals should require authors to include dedicated supplementary methods detailing antibody validation for each application. These collective practices would significantly enhance data reliability and cross-study comparability, accelerating progress in understanding SLC30A9's biological functions and disease implications while reducing research waste from unreproducible antibody-based experiments.