SLC2A9 Antibody

What is SLC2A9 and why is it significant in research?

SLC2A9 (solute carrier family 2 member 9), also known as GLUT9, is a facilitative glucose transporter that functions as a high-capacity urate transporter in humans. It has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney . The significance of SLC2A9 in research stems from its association with serum uric acid levels, which correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease . Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate levels and gout, making it an important target for researchers investigating these conditions .

What applications are SLC2A9 antibodies commonly used for?

SLC2A9 antibodies are versatile tools used across multiple experimental applications:

These applications allow researchers to detect, quantify, and localize SLC2A9 protein in various experimental contexts .

How should SLC2A9 antibodies be stored and handled to maintain optimal activity?

For optimal antibody performance, SLC2A9 antibodies should be stored at -20°C where they remain stable for one year after shipment . The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, simplifying laboratory workflow . When working with smaller quantities (e.g., 20μl sizes), be aware that these may contain 0.1% BSA as a stabilizer . Always avoid repeated freeze-thaw cycles, which can degrade antibody quality and compromise experimental results.

What is the molecular weight of SLC2A9 protein and how does this affect antibody selection?

The calculated molecular weight of SLC2A9 is 59 kDa based on its 540 amino acid sequence, while the observed molecular weight in experimental settings typically ranges from 56-59 kDa . This slight variation may reflect post-translational modifications or splice variant differences. When selecting an antibody, researchers should verify that the antibody recognizes the correct molecular weight band in Western blots. Additionally, researchers should consider whether their experiment requires an antibody that recognizes specific splice variants of SLC2A9, as there are two main variants with differential expression patterns in tissues .

How can SLC2A9 antibodies be used to investigate the rs16890979 SNP's effect on uric acid metabolism?

The rs16890979 SNP in SLC2A9 has been shown to reduce uric acid (UA) absorption in human kidney organoids . To investigate this SNP's effects, researchers can employ the following methodological approach:

This integrated approach allows researchers to establish direct causal relationships between the rs16890979 SNP and altered uric acid handling, while controlling for genetic background variables that might confound results in human population studies .

What controls should be included when using SLC2A9 antibodies in flow cytometry experiments?

When using SLC2A9 antibodies in flow cytometry, comprehensive controls are essential for result validation:

• Isotype control: Include an appropriate isotype-matched control antibody (e.g., rabbit IgG at the same concentration as the primary antibody) to establish baseline fluorescence and assess non-specific binding .

• Unlabelled sample control: Process cells without primary and secondary antibody incubation to determine autofluorescence levels .

• Secondary antibody-only control: Include samples treated only with the secondary antibody to evaluate non-specific binding of the secondary antibody.

• Positive control: Use cell lines known to express SLC2A9 (e.g., U937 cells) to validate antibody performance .

• Negative control: Include SLC2A9 knockdown samples using targeted siRNA to confirm antibody specificity .

• Permeabilization controls: Since SLC2A9 is a transmembrane protein, compare permeabilized and non-permeabilized samples to distinguish between surface and total cellular expression.

For optimal results, cells should be fixed with 4% paraformaldehyde, permeabilized with appropriate buffer, and blocked with 10% normal goat serum before incubation with the SLC2A9 antibody . Secondary antibody selection should match the host species of the primary (e.g., DyLight®488 conjugated goat anti-rabbit IgG) .

How can researchers troubleshoot inconsistent SLC2A9 antibody staining patterns in tissue samples?

• Optimize antigen retrieval: SLC2A9 antibody staining in tissues may require specific antigen retrieval methods. Use TE buffer at pH 9.0 as the primary method, with citrate buffer at pH 6.0 as an alternative approach when results are suboptimal .

• Validate antibody specificity: Confirm antibody specificity using positive and negative controls. SLC2A9 antibodies should show strong reactivity in tissues known to express the protein, such as liver, kidney, and specific cell lines (SMMC-7721, HepG2, L02) .

• Titrate antibody concentration: Perform a dilution series to determine the optimal antibody concentration. For immunohistochemistry, recommended dilutions range from 1:500 to 1:2000, but the optimal concentration may vary by tissue type and preparation method .

• Cross-validate with alternative detection methods: Confirm staining patterns using multiple detection methods (IHC, IF, WB) or alternative antibodies targeting different epitopes of SLC2A9 .

• Consider splice variant expression: SLC2A9 has two main splice variants with potentially different tissue distribution patterns. Ensure your antibody can detect the specific variant(s) relevant to your research question .

• Assess fixation effects: Compare results across different fixation protocols, as overfixation can mask epitopes while underfixation may compromise tissue morphology.

How can SLC2A9 antibodies be used to investigate the relationship between SLC2A9 expression and epithelial-mesenchymal transition (EMT) in kidney injury?

Recent research has revealed a potential link between SLC2A9 function, uric acid levels, and epithelial-mesenchymal transition (EMT) in kidney injury . To investigate this relationship using SLC2A9 antibodies:

• Generate experimental models: Create kidney organoids with wild-type SLC2A9, SLC2A9 overexpression (OE), and SLC2A9 knockdown using lentiviral vectors .

• Validate expression levels: Use Western blotting with SLC2A9 antibodies to confirm successful modulation of protein expression .

• Uric acid challenge studies: Treat organoids with varying concentrations of uric acid and assess cellular responses .

• Multi-parameter immunofluorescence: Employ co-staining protocols using SLC2A9 antibodies alongside markers of EMT (e.g., E-cadherin, vimentin, α-SMA) to visualize and quantify EMT changes in relation to SLC2A9 expression and localization .

• Quantitative image analysis: Apply digital image analysis to quantify changes in SLC2A9 expression, subcellular localization, and correlation with EMT markers.

• Functional correlation: Correlate observed changes in SLC2A9 distribution with uric acid uptake measurements to establish functional relationships.

This approach allows researchers to assess whether SLC2A9 expression levels directly influence EMT processes during hyperuricemic conditions, potentially revealing mechanisms of renal injury in conditions like gout and metabolic syndrome .

What are the optimal conditions for using SLC2A9 antibodies in immunofluorescence applications?

For optimal immunofluorescence results with SLC2A9 antibodies:

• Sample preparation: For cell lines such as A549, use enzyme antigen retrieval methods (e.g., IHC enzyme antigen retrieval reagent) with a 15-minute incubation period .

• Blocking: Block non-specific binding sites with 10% goat serum (match blocking serum to the host species of your secondary antibody) .

• Primary antibody incubation: Use SLC2A9 antibody at a concentration of 2μg/mL and incubate overnight at 4°C for optimal signal-to-noise ratio .

• Secondary antibody selection: Use fluorophore-conjugated secondary antibodies (e.g., DyLight®488 conjugated Goat Anti-Rabbit IgG) at a 1:100 dilution with a 30-minute incubation at 37°C .

• Nuclear counterstaining: Counterstain with DAPI to visualize nuclei and provide cellular context for SLC2A9 localization .

• Visualization parameters: Use appropriate filter sets for the selected fluorophores and adjust exposure settings to prevent photobleaching while maintaining adequate signal intensity .

• Controls: Include secondary-only controls and, where possible, samples with validated high and low SLC2A9 expression to confirm specificity .

These conditions should be optimized for each specific tissue or cell type under investigation.

How can researchers effectively validate SLC2A9 antibody specificity for their experimental systems?

Thorough validation of antibody specificity is crucial for generating reliable research data. For SLC2A9 antibodies:

• Genetic knockdown/knockout validation: Use siRNA knockdown or CRISPR/Cas9 knockout of SLC2A9 to create negative control samples that should show reduced or absent antibody signal .

• Overexpression systems: Compare signal intensity between wild-type samples and samples overexpressing SLC2A9 using viral vectors or transfection .

• Western blot analysis: Confirm single band detection at the expected molecular weight (56-59 kDa) in positive control samples and absence/reduction in knockdown samples .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples; this should eliminate specific binding.

• Cross-species reactivity testing: Validate the antibody in samples from multiple species if cross-species applications are planned. Current SLC2A9 antibodies show reactivity across human, mouse, rat, rabbit, and pig samples .

• Multiple antibody comparison: Use antibodies from different sources or targeting different epitopes and compare staining patterns.

• Correlation with mRNA expression: Compare antibody staining intensity with SLC2A9 mRNA levels as measured by qRT-PCR to confirm expression pattern consistency .

What approaches can be used to study both SLC2A9 splice variants simultaneously with antibodies?

SLC2A9 exists in two main splice variants with distinct expression patterns and potentially different functions . To study both variants:

• Select appropriate antibodies: Choose antibodies raised against epitopes common to both variants or use two separate antibodies specific to each variant. For instance, some researchers have developed polyclonal antibodies against N-terminal peptides of both splice variants .

• Western blot analysis: Use gradient gels with high resolution in the 50-60 kDa range to potentially separate the slightly different molecular weights of the two variants.

• Isoform-specific controls: Create expression vectors for each splice variant individually as positive controls for antibody validation.

• Two-color immunofluorescence: If using isoform-specific primary antibodies from different host species, implement two-color immunofluorescence to visualize differential localization patterns.

• RT-PCR validation: Correlate antibody detection with RT-PCR using primers specific to each splice variant to confirm expression patterns.

• Mass spectrometry validation: Use immunoprecipitation with the SLC2A9 antibody followed by mass spectrometry to identify which isoforms are being detected.

• Tissue-specific controls: Include tissues known to differentially express the splice variants (e.g., kidney proximal tubule for variant-specific expression) .

This multi-faceted approach allows researchers to distinguish between the variants and understand their potentially distinct roles in urate transport and disease processes.

How should researchers interpret changes in SLC2A9 expression in relation to uric acid handling?

When interpreting changes in SLC2A9 expression detected by antibodies in relation to uric acid handling:

• Functional correlation: Research has established that SLC2A9 is responsible for uric acid (UA) absorption, with increased expression leading to greater UA uptake and decreased expression reducing uptake . Changes in expression should be interpreted in this functional context.

• Genetic modification effects: The rs16890979 SNP reduces UA absorption despite normal SLC2A9 expression levels, indicating that protein function, not just expression level, is critical to consider .

• Quantification approaches: Use densitometry for Western blots, mean fluorescence intensity for flow cytometry, and digital image analysis for IHC/IF to quantify expression changes objectively .

• Threshold determination: Establish biologically significant thresholds for expression changes based on functional assays of urate transport, as small changes in expression might not translate to meaningful functional differences .

• Splice variant consideration: Consider differential regulation of the two SLC2A9 splice variants, as their relative expression may change in disease states or experimental conditions .

• Subcellular localization: Assess changes in membrane localization, not just total protein expression, as functional transport requires proper membrane integration .

• Correlation with pathology: Relate SLC2A9 expression changes to markers of renal injury or EMT to establish clinically relevant relationships .

What experimental design is recommended for investigating SLC2A9 function in kidney organoid models using antibodies?

For robust investigation of SLC2A9 function in kidney organoid models:

• Genetic manipulation strategy: Implement a comprehensive genetic approach including:

  • Wild-type controls

  • SLC2A9 overexpression models

  • SLC2A9 knockdown models

  • Specific SNP variants (e.g., rs16890979 in both heterozygous and homozygous forms)

• Expression verification: Use qRT-PCR for mRNA levels and Western blotting with SLC2A9 antibodies to confirm protein expression levels in each model .

• Developmental characterization: Apply SLC2A9 antibodies in immunofluorescence at multiple time points during organoid development to track expression patterns and localization changes .

• Functional assays: Implement uric acid uptake assays in conjunction with antibody-based protein detection to correlate expression with function .

• Challenge experiments: Expose organoids to varying uric acid concentrations and use antibodies to track changes in SLC2A9 expression and localization in response to challenges .

• Co-localization studies: Perform dual staining with SLC2A9 antibodies and markers of specific kidney structures to confirm appropriate anatomical expression .

• Pathological modeling: Use antibodies to assess SLC2A9 changes in organoid models of kidney disease states, correlating with functional and structural changes .

This comprehensive approach allows for detailed investigation of SLC2A9's role in normal physiology and disease states using the organoid model system .

How can contradictory results between SLC2A9 antibody staining and functional assays be reconciled?

When faced with discrepancies between antibody staining and functional assay results:

• Epitope accessibility assessment: Determine if the antibody's epitope might be masked by protein conformation changes, post-translational modifications, or protein-protein interactions that occur during different functional states.

• Functional vs. non-functional protein: Consider that antibodies may detect both functional and non-functional forms of SLC2A9, while functional assays only measure active protein. The rs16890979 SNP exemplifies this scenario, showing normal protein expression but reduced function .

• Splice variant differential detection: Verify whether your antibody detects both SLC2A9 splice variants equally, as functional differences between variants might explain discrepancies .

• Subcellular localization analysis: Perform subcellular fractionation followed by Western blotting or high-resolution imaging to determine if the protein is correctly localized to the membrane for function .

• Post-translational modification examination: Investigate whether post-translational modifications affect function but not antibody recognition. Consider phosphorylation-specific antibodies if appropriate.

• Co-factor dependency: Assess whether SLC2A9 function depends on co-factors or interacting proteins not present in all experimental conditions.

• Temporal considerations: Implement time-course experiments to determine if expression precedes function or vice versa, explaining apparent contradictions at single time points.

By systematically addressing these possibilities, researchers can reconcile contradictory results and gain deeper insights into SLC2A9 biology.

How can SLC2A9 antibodies be used in multi-omics approaches to understand urate transport mechanisms?

Integrating SLC2A9 antibody-based techniques into multi-omics research approaches offers powerful insights:

• Proteomics integration: Use immunoprecipitation with SLC2A9 antibodies followed by mass spectrometry to identify protein interaction partners that may regulate urate transport .

• Phosphoproteomics: Apply phospho-specific antibodies or general SLC2A9 antibodies in immunoprecipitation followed by phosphoproteomic analysis to identify regulatory phosphorylation sites.

• Genomics correlation: Correlate antibody-detected protein expression with genomic data on SLC2A9 variants (e.g., rs16890979) to understand genotype-phenotype relationships .

• Transcriptomics validation: Use SLC2A9 antibodies to confirm protein-level changes predicted by transcriptomic analyses, addressing post-transcriptional regulation questions.

• Spatial transcriptomics correlation: Combine immunofluorescence using SLC2A9 antibodies with spatial transcriptomics to map expression patterns in tissue context.

• Metabolomics integration: Correlate SLC2A9 protein levels detected by antibodies with metabolomic profiles, particularly uric acid and related metabolites.

• Single-cell approaches: Apply SLC2A9 antibodies in single-cell proteomics or CyTOF to understand cell-to-cell variability in expression and correlation with cell state or function.

This integrated approach allows researchers to build comprehensive models of urate transport regulation that span from genetic variation to functional outcomes.

What are the considerations for using SLC2A9 antibodies in studying the link between uric acid transport and metabolic disorders?

When investigating links between SLC2A9, uric acid transport, and metabolic disorders:

• Model selection: Choose appropriate models that recapitulate key aspects of metabolic disorders, such as kidney organoids exposed to hyperglycemic conditions or animal models of metabolic syndrome .

• Tissue specificity: Apply SLC2A9 antibodies to multiple relevant tissues beyond kidney, including liver and adipose tissue, as SLC2A9 may have different roles in different metabolic tissues .

• Diet and environmental factors: Design experiments that account for dietary factors that influence uric acid levels, and use SLC2A9 antibodies to assess protein response to these factors.

• Inflammation correlation: Perform co-staining with SLC2A9 antibodies and markers of inflammation to assess relationships between urate transport, inflammation, and metabolic dysfunction.

• Co-transporter analysis: Study SLC2A9 in conjunction with other transporters and metabolic enzymes to build a systems-level understanding of urate handling in metabolic disorders.

• Sex-specific differences: Design studies to capture sex-specific differences in SLC2A9 expression and function, as metabolic disorders often show sexual dimorphism.

• Therapeutic interventions: Use SLC2A9 antibodies to assess protein expression changes in response to therapeutic interventions targeting metabolic disorders or hyperuricemia.

These considerations enable researchers to establish mechanistic links between SLC2A9 function and the pathophysiology of metabolic disorders that may inform therapeutic approaches.