SLC12A3 Antibody, HRP conjugated

What is SLC12A3 and what is its functional significance in research models?

SLC12A3 (Solute carrier family 12 member 3) functions as an electroneutral sodium and chloride ion cotransporter that plays a crucial role in mediating sodium and chloride reabsorption in kidney distal convoluted tubules . Research has demonstrated that SLC12A3, also known as NCC (Na-Cl cotransporter), TSC, or thiazide-sensitive sodium-chloride cotransporter, additionally acts as a receptor for the pro-inflammatory cytokine IL18, contributing to cytokine production including IFNG, IL6, IL18, and CCL2 . The protein may function either independently of IL18R1 or in a complex with it, making it relevant for both renal physiology and inflammatory response studies . When designing experiments targeting SLC12A3, researchers should consider its predominant expression in distal convoluted tubules and its involvement in both sodium homeostasis and inflammatory signaling pathways.

What experimental applications are SLC12A3 antibodies validated for?

Based on validation studies, commercially available SLC12A3 antibodies have been successfully employed in multiple experimental applications. Western blotting (WB) and immunohistochemistry on paraffin-embedded sections (IHC-P) represent the primary validated applications . When selecting an SLC12A3 antibody for your research, confirmation of species reactivity is essential as most antibodies demonstrate reactivity with human and pig samples, with potential cross-reactivity with recombinant human SLC12A3 protein . For Western blotting applications, a standard working concentration of 1 μg/mL has been established as optimal through validation studies, though optimization for specific experimental conditions may be necessary .

How should sample preparation be optimized when working with SLC12A3 antibodies?

For optimal detection of SLC12A3 using antibodies, sample preparation protocols should be tailored to the experimental application. For protein extraction prior to Western blotting, research indicates that RIPA buffer supplemented with 1% PMSF provides effective protein isolation . Following protein denaturation, separation on 12% SDS-PAGE and transfer to polyvinylidene fluoride membranes has been established as an effective protocol . When working with tissue samples for IHC-P, standard paraffin embedding protocols are compatible with SLC12A3 detection, though antigen retrieval methods may need optimization based on fixation conditions. For cell-based experiments, HEK293T cells have been successfully utilized as expression systems for both wild-type and mutant SLC12A3 proteins .

What strategies can improve detection specificity when using HRP-conjugated SLC12A3 antibodies?

To maximize detection specificity with HRP-conjugated SLC12A3 antibodies, multiple optimization approaches should be considered. First, implement a tiered blocking strategy using 3-5% BSA in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1-2 hours at room temperature before antibody incubation . Second, optimize primary antibody concentration through serial dilution experiments (typically between 0.5-2 μg/mL) and extend incubation times at 4°C overnight to improve specific binding while reducing background . If using secondary detection systems, HRP-conjugated secondary antibodies at 1:20000 dilution have shown optimal signal-to-noise ratios in published protocols . For critical experiments, inclusion of absorption controls where the antibody is pre-incubated with recombinant SLC12A3 peptide can confirm binding specificity. Additionally, signal detection optimization using enhanced chemiluminescence with varying exposure times will help capture specific signals while minimizing background.

How can researchers accurately evaluate SLC12A3 expression in the context of genetic variants?

When studying SLC12A3 genetic variants, a comprehensive approach combining genetic and protein analysis is essential. Recent research demonstrates that variants like p.E240K and p.L892P can significantly affect protein expression levels . For accurate evaluation, implement the following protocol:

• Confirm variant sequences using Sanger sequencing following PCR amplification of the target region .

• Quantify mRNA expression levels using qPCR with primers targeting conserved regions (forward: 5′-CAAGGATGACGATGACAAGC-3′, reverse: 5′-TCGTGTTGTAGCCAAAGGTG-3′) and normalize to appropriate housekeeping genes (e.g., actin) .

• Assess protein expression through Western blotting using validated SLC12A3 antibodies (1:1000 dilution) with GAPDH (1:1000) as loading control .

• Evaluate subcellular localization through immunofluorescence microscopy to determine if variants affect trafficking.

• Conduct functional assays such as Na+ uptake experiments to correlate expression changes with functional impacts .

Research has shown that some variants (like p.L892P) can affect both expression levels and functional activity, while others (like p.E240K) primarily impact protein expression without affecting localization .

What experimental design considerations are essential when using SLC12A3 antibodies to study Gitelman syndrome?

Gitelman syndrome (GS) research using SLC12A3 antibodies requires specific experimental considerations due to the disease's molecular basis. First, establish appropriate control groups including wild-type samples, heterozygous carriers (who may show biochemical abnormalities), and GS patients with known pathogenic variants . Research indicates that compound heterozygous mutations in SLC12A3 typically cause more severe phenotypes, necessitating genotype-phenotype correlation studies .

For comprehensive analysis, combine the following methodological approaches:

• Genetic screening through whole-exome sequencing or targeted sequencing of SLC12A3 .

• Bioinformatic analysis of identified variants using multiple prediction tools (SIFT, PolyPhen-2, MutationTaster) .

• Structural modeling of mutant proteins using SWISS-MODEL to predict impacts on protein structure .

• Functional validation through:

  • Expression studies in heterologous systems (HEK293T cells)

  • Sodium uptake assays to evaluate transporter function

  • Membrane localization studies to assess trafficking

Recent research has demonstrated that some variants predominantly affect protein expression (p.E240K) while others may impact both expression and function (p.L892P), highlighting the importance of comprehensive functional assessment .

How can non-specific binding be reduced when using SLC12A3 antibodies in complex tissue samples?

Non-specific binding represents a significant challenge when working with SLC12A3 antibodies in complex tissues like kidney. To minimize this issue, implement the following optimized protocol based on research findings:

• Tissue preparation: Use freshly prepared 4% paraformaldehyde fixation limited to 24 hours and implement a graded ethanol dehydration protocol to preserve epitope accessibility.

• Antigen retrieval: Perform heat-induced epitope retrieval using 10mM citrate buffer (pH 6.0) for 20 minutes at 95°C, as this has been shown to maximize SLC12A3 detection while minimizing background.

• Blocking optimization: Implement a dual blocking approach with 5% normal serum (matched to secondary antibody host) for 1 hour followed by 2% BSA with 0.1% Triton X-100 for 30 minutes.

• Primary antibody dilution: Optimize through serial dilutions (typically between 1:500-1:2000), with overnight incubation at 4°C.

• Washing protocol: Extend washing steps to 4×10 minutes with PBS containing 0.1% Tween-20 to remove unbound antibody.

• Absorption controls: Include controls where antibody is pre-incubated with recombinant SLC12A3 protein to confirm binding specificity.

This comprehensive approach has been shown to substantially reduce non-specific binding while maintaining detection sensitivity for SLC12A3 in renal tissue sections.

What are the critical variables affecting sodium uptake assays when evaluating SLC12A3 function?

Sodium uptake assays represent a critical functional assessment for SLC12A3 research. Research findings have identified several variables that significantly impact assay performance and data interpretation:

Recent functional studies investigating SLC12A3 mutations demonstrated that both p.E240K and p.L892P variants significantly reduced Na+ uptake activity, with p.L892P showing more pronounced effects (>70% reduction compared to wild-type) . These findings highlight the importance of including appropriate wild-type and known mutant controls when evaluating novel variants.

How can researchers differentiate between expression and functional effects of SLC12A3 mutations?

Distinguishing between mutations that affect protein expression versus function requires a methodical approach combining multiple experimental techniques. Based on recent research investigating novel SLC12A3 variants, implement the following integrated protocol:

• mRNA expression analysis: Quantify transcripts using RT-qPCR to determine if mutations affect transcript stability. Research shows some mutations like p.L892P significantly reduce mRNA levels .

• Protein expression assessment: Perform Western blotting with validated SLC12A3 antibodies to quantify total protein levels. Studies have demonstrated that both p.E240K and p.L892P variants significantly reduce protein expression compared to wild-type .

• Membrane localization studies: Conduct immunofluorescence microscopy to determine if mutant proteins reach the plasma membrane. Research indicates some mutations affect trafficking while others (like p.E240K and p.L892P) may reach the membrane but with reduced expression .

• Functional analysis: Perform sodium uptake assays to measure transporter activity. Normalizing functional data to expression levels can distinguish between:

  • Mutations primarily affecting expression (functional activity correlates with expression levels)

  • Mutations affecting intrinsic function (activity reduction exceeds expression reduction)

This comprehensive approach revealed that p.E240K primarily reduces expression while maintaining relative function per molecule, whereas p.L892P affects both expression and intrinsic function of each transporter molecule .

How can protein structure prediction tools enhance understanding of SLC12A3 antibody epitope accessibility?

Protein structure prediction represents a valuable approach for optimizing antibody selection and experimental design when working with SLC12A3. Implementation of computational tools like SWISS-MODEL and PyMOL Viewer can generate three-dimensional structures of wild-type and mutant SLC12A3 proteins . These models provide critical insights into epitope accessibility for antibody binding.

For optimal epitope analysis, researchers should:

• Generate SLC12A3 structural models using homology modeling against related transporters.

• Analyze surface-exposed regions using solvent accessibility calculations to identify potential epitope regions.

• Evaluate the impact of mutations on protein secondary and tertiary structure, particularly for regions targeted by antibodies .

• Consider the effects of post-translational modifications on epitope recognition.

Research investigating SLC12A3 mutations has demonstrated that structural alterations can significantly affect protein conformation. For example, the p.E240K mutation, located near the third large extracellular loop, and p.L892P, located in the intracellular C-terminal domain, both alter protein secondary structure and stability . These structural changes may impact antibody binding efficiency, highlighting the importance of selecting antibodies targeting regions less affected by common mutations.

What considerations are important when developing multiplexed detection systems involving SLC12A3?

Multiplexed detection systems can provide comprehensive insights into SLC12A3 biology by simultaneously evaluating expression, localization, and co-localization with interaction partners. When developing such systems, several critical factors must be considered:

• Antibody compatibility: Select primary antibodies raised in different host species to enable simultaneous detection. For SLC12A3, rabbit polyclonal antibodies have shown reliable detection .

• Signal separation: When using multiple fluorophores or enzyme systems, ensure sufficient spectral separation or use sequential detection protocols to prevent cross-reactivity.

• Optimization of detection systems: For HRP-conjugated antibodies in multiplexed systems, consider using:

  • Tyramide signal amplification for enhanced sensitivity

  • Different substrates with distinct colorimetric or chemiluminescent properties

  • Sequential detection with HRP inactivation between rounds

• Validation controls: Include single-staining controls and absorption controls to confirm specificity in the multiplexed context.

• Quantification considerations: Develop standardized image analysis protocols that account for potential signal overlap and variability in background.

Multiplexed approaches are particularly valuable when studying SLC12A3 in the context of Gitelman syndrome, where correlation between transporter expression, localization, and interacting proteins like kinases involved in phosphorylation may provide mechanistic insights into pathogenesis.

How might emerging antibody engineering techniques improve SLC12A3 detection specificity and sensitivity?

Emerging antibody engineering technologies offer promising approaches to enhance SLC12A3 detection. Several technological advancements warrant consideration:

• Single-chain variable fragment (scFv) development targeting specific SLC12A3 epitopes may improve tissue penetration and reduce background compared to conventional antibodies.

• Recombinant monoclonal antibody production against highly conserved epitopes could enhance detection consistency across species models.

• Site-specific conjugation technologies that control the HRP:antibody ratio and conjugation site can significantly improve signal-to-noise ratios compared to traditional random conjugation methods.

• Nanobody development (single-domain antibodies) may provide access to epitopes in complex three-dimensional conformations of SLC12A3 that are inaccessible to conventional antibodies.

• Proximity-based detection systems combining SLC12A3 antibodies with complementary oligonucleotides for signal amplification could dramatically enhance sensitivity for detecting low expression levels.

Future research should focus on developing these technologies specifically for SLC12A3 detection, particularly for applications requiring quantitative analysis of expression in the context of genetic variants associated with Gitelman syndrome.

What methodological approaches can integrate SLC12A3 protein studies with functional genomics?

Integrating SLC12A3 protein studies with functional genomics requires methodological innovations that connect genetic variants to protein function. Based on recent research approaches, consider implementing:

• CRISPR/Cas9 gene editing to introduce specific SLC12A3 variants in cellular or animal models, followed by antibody-based detection to assess expression patterns and levels .

• Correlation of genetic variant data from whole-exome sequencing with protein expression patterns quantified through standardized immunodetection protocols .

• Development of high-throughput screening systems that combine variant generation with automated immunodetection and functional assays.

• Integration of transcriptomic data with protein-level quantification to identify potential regulatory mechanisms affecting SLC12A3 expression.

• Implementation of systems biology approaches that correlate SLC12A3 variant effects across genomic, transcriptomic, and proteomic levels.

Recent research has successfully applied several of these approaches to characterize novel SLC12A3 variants, demonstrating that integrating genetic analysis with protein-level studies can provide comprehensive insights into transporter pathophysiology in conditions like Gitelman syndrome .