SLC12A3 Antibody

What is SLC12A3 and why is it important in kidney research?

SLC12A3 (Solute Carrier Family 12 Member 3) functions as an electroneutral sodium and chloride ion cotransporter that serves as a key mediator of sodium and chloride reabsorption in kidney distal convoluted tubules. This protein significantly influences electrolyte and fluid balance, making it crucial for understanding renal physiology and pathophysiology . Recent evidence also suggests SLC12A3 may act as a receptor for the pro-inflammatory cytokine IL18, contributing to cytokine production including IFNG, IL6, IL18, and CCL2 . The protein is predominantly expressed in the kidney and has been implicated in disorders such as Gitelman Syndrome, characterized by hypokalemia and metabolic alkalosis due to SLC12A3 mutations . Understanding SLC12A3 function has applications across nephrology, cardiovascular research, and inflammatory disease studies.

What applications are SLC12A3 antibodies commonly used for in research settings?

SLC12A3 antibodies can be utilized across multiple experimental applications depending on specific research needs:

The selection of application should be based on experimental goals, whether studying protein expression levels, cellular localization, or protein-protein interactions .

How do I select the appropriate epitope region when choosing an SLC12A3 antibody?

Selection of the appropriate epitope region is crucial for experimental success and depends on your specific research question. Available SLC12A3 antibodies target various amino acid regions including:

• N-terminal regions (AA 3-146, AA 74-95)

• Central regions

• C-terminal regions (AA 867-1024, AA 951-1021)

Each epitope region offers distinct advantages:

• N-terminal antibodies (e.g., AA 74-95) are useful for detecting the full-length protein and are less affected by C-terminal post-translational modifications .

• C-terminal antibodies may better detect regulatory changes as this region contains important phosphorylation sites that affect NCC activity.

• For mutation studies, select antibodies targeting regions unaffected by your mutation of interest. For example, when studying the p.E240K and p.L892P variants, antibodies targeting other regions would be preferable for detecting expression differences .

Always evaluate the conservation of your target epitope across species if planning cross-species studies, as amino acid variations may affect antibody recognition .

What factors should be considered when interpreting SLC12A3 antibody Western blot results?

When interpreting Western blot results with SLC12A3 antibodies, several critical factors must be considered:

• Expected molecular weight: SLC12A3 typically appears at ~160 kDa, but glycosylation and other post-translational modifications can alter migration patterns . Multiple bands may represent different glycosylation states rather than non-specific binding.

• Sample preparation: Membrane protein extraction methods significantly impact detection quality. Use specialized membrane protein extraction buffers and avoid excessive heating which can cause protein aggregation.

• Cross-reactivity assessment: Many SLC12A3 antibodies demonstrate cross-reactivity with human, mouse, rat, and dog samples . Validate specificity with appropriate controls, particularly when working with less common species.

• Loading controls: For kidney tissue samples, consider using multiple loading controls as traditional housekeeping proteins may vary in expression across different renal segments.

• Quantification limitations: When comparing wild-type and mutant SLC12A3 variants, recognize that decreased signal intensity could reflect altered epitope accessibility rather than reduced expression .

A systematic troubleshooting approach is essential when unexpected bands appear or expected signals are absent.

How can SLC12A3 antibodies be optimized for detecting variant proteins in Gitelman Syndrome research?

Optimizing SLC12A3 antibody protocols for detecting variant proteins in Gitelman Syndrome research requires sophisticated approaches:

• Variant-specific considerations: When studying specific variants like p.E240K and p.L892P, select antibodies targeting unaffected epitopes. Recent research demonstrates that these variants significantly reduce protein expression and Na+ transport activity, with p.L892P showing more severe effects .

• Complementary techniques: Combine protein detection methods with functional assays:

  • Quantitative RT-PCR to assess transcript levels (using validated primers such as forward 5′-CAAGGATGACGATGACAAGC-3′, reverse 5′-TCGTGTTGTAGCCAAAGGTG-3′)

  • Cell surface biotinylation to specifically quantify membrane-expressed NCC

  • Sodium uptake assays to correlate protein expression with functional activity

• Expression system optimization: When expressing mutant constructs in heterologous systems, consider:

  • Codon optimization for the expression system

  • Temperature-sensitive folding rescue (30°C incubation)

  • Proteasome inhibitors to assess degradation pathways

• Structural impact analysis: Integrate antibody detection with structural modeling to correlate epitope accessibility with predicted conformational changes in variant proteins .

These approaches allow for comprehensive characterization of how mutations affect protein expression, trafficking, and function in Gitelman Syndrome research.

What are the best practices for using SLC12A3 antibodies in multiplex immunofluorescence studies?

Implementing multiplex immunofluorescence with SLC12A3 antibodies requires careful planning and execution to avoid cross-reactivity and ensure reliable co-localization data:

• Antibody panel design:

  • Select SLC12A3 antibodies from different host species than other target antibodies

  • If using multiple rabbit antibodies, consider sequential immunostaining with HRP inactivation between rounds

  • Validate each antibody individually before multiplexing

• Fluorophore selection and spectral considerations:

  • SLC12A3 antibodies are available with various conjugates including FITC, making them versatile for multiplex designs

  • Choose fluorophores with minimal spectral overlap

  • Include appropriately matched negative controls for each fluorophore

• Tissue-specific optimization:

  • For kidney sections, optimize antigen retrieval conditions that work for all target proteins

  • Consider tissue autofluorescence, particularly in kidney samples containing lipofuscin

• Image acquisition and analysis:

  • Use spectral unmixing for closely related fluorophores

  • Perform quantitative colocalization analysis using appropriate software

  • Report Pearson's or Mander's coefficients when claiming colocalization

Research has successfully employed multiplex approaches to co-localize SLC12A3 with other renal transporters and regulatory proteins, enabling sophisticated studies of transporter regulation and trafficking .

How do post-translational modifications of SLC12A3 affect antibody recognition and experimental design?

Post-translational modifications (PTMs) of SLC12A3 significantly impact antibody recognition and necessitate careful experimental design:

• Phosphorylation effects:

  • SLC12A3 is phosphorylated in response to IL18 stimulation, which may mask certain epitopes

  • Phospho-specific antibodies can be used to monitor activation state

  • For studying phosphorylation-dependent events, include phosphatase inhibitors in extraction buffers

• Ubiquitination considerations:

  • SLC12A3 undergoes ubiquitination essential for regulation of endocytosis

  • Ubiquitinated forms may migrate at higher molecular weights than expected

  • For studying ubiquitination, use deubiquitinase inhibitors and consider immunoprecipitation followed by ubiquitin detection

• Glycosylation impact:

  • N-glycosylation affects SLC12A3 maturation and membrane trafficking

  • Deglycosylation treatments (PNGase F) can confirm glycosylation status and explain molecular weight variations

  • Antibodies targeting heavily glycosylated regions may show reduced affinity

• Experimental approach selection based on PTM research questions:

  • To study basal protein levels: use antibodies against stable, minimally modified regions

  • To investigate regulatory mechanisms: select antibodies unaffected by the specific PTM of interest

  • For comprehensive PTM analysis: combine targeted antibodies with mass spectrometry techniques

Understanding the interplay between these modifications is crucial when investigating the molecular mechanisms underlying SLC12A3 dysfunction in pathological conditions .

What methodological approaches can resolve contradictory results when using different SLC12A3 antibodies?

Contradictory results with different SLC12A3 antibodies are common challenges in research. Systematic resolution strategies include:

• Comprehensive epitope mapping:

  • Document the exact epitope region for each antibody (e.g., AA 74-95 vs. AA 867-1024)

  • Assess epitope accessibility in different experimental conditions

  • Consider conformational vs. linear epitope recognition patterns

• Validation hierarchy implementation:

  • Genetic approaches: siRNA/CRISPR knockdown to confirm specificity

  • Recombinant protein controls with tagged constructs

  • Peptide competition assays to confirm epitope specificity

  • Cross-validation with non-antibody-based detection methods

• Sample preparation standardization:

  • Develop a standardized tissue/cell processing protocol

  • Compare different fixation methods systematically

  • Evaluate membrane protein extraction techniques side-by-side

• Antibody performance documentation:

  Antibody Type	Optimal Applications	Known Limitations	Recommended Controls
  Polyclonal (AA 74-95)	WB, IHC, ICC, IEM, IF	Batch variation	Peptide blocking
  Monoclonal [EPR27106-48]	Dot, IHC-P, WB	Limited epitope recognition	Knockout tissue
  Recombinant antibodies	Highly reproducible detection	May have narrower reactivity	Tagged recombinant protein

• Application-specific optimization:

  • For WB: Optimize protein extraction, denaturation, and transfer conditions

  • For IHC/IF: Compare different antigen retrieval methods and detection systems

How can SLC12A3 antibodies be effectively used to investigate protein-protein interactions in renal physiology?

Leveraging SLC12A3 antibodies for protein-protein interaction studies requires specialized approaches:

• Co-immunoprecipitation optimization:

  • Select antibodies with minimal interference with interaction domains

  • Use membrane-compatible lysis buffers to maintain native conformation

  • Consider crosslinking approaches for transient interactions

  • Research has demonstrated SLC12A3 interactions with KLHL3 and IL18R1 using such approaches

• Proximity ligation assay (PLA) implementation:

  • Combine SLC12A3 antibodies with antibodies against putative interacting partners

  • Optimize primary antibody concentrations to minimize background

  • Include appropriate negative controls (known non-interacting proteins)

  • This technique can visualize interactions in situ within renal tissue

• FRET/BRET-based approaches:

  • Use antibodies to validate interaction results from fluorescence/bioluminescence resonance energy transfer

  • Compare endogenous interactions (antibody-detected) with overexpression systems

• Mass spectrometry validation:

  • Optimize immunoprecipitation conditions for downstream mass spectrometry

  • Use crosslinking mass spectrometry to map interaction interfaces

  • Validate novel interactions with reciprocal co-immunoprecipitation using SLC12A3 antibodies

• Physiological relevance confirmation:

  • Investigate interactions under various physiological stimuli (e.g., IL18 treatment increases SLC12A3-IL18R1 interaction)

  • Assess interaction changes in disease models or with variant proteins

These methodologies provide complementary approaches to elucidate SLC12A3's role in protein complexes that regulate renal sodium handling and inflammatory signaling .

What are the latest methodological advances in using SLC12A3 antibodies for studying transporter trafficking defects?

Recent methodological advances have enhanced the utility of SLC12A3 antibodies for investigating trafficking defects:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM/PALM) with SLC12A3 antibodies enables visualization of nanoscale distribution in the distal convoluted tubule

  • Live-cell imaging with membrane-impermeant antibodies against extracellular epitopes can track surface dynamics

  • Correlative light and electron microscopy (CLEM) combines immunofluorescence with ultrastructural localization

• Quantitative surface expression analysis:

  • Cell surface biotinylation combined with SLC12A3 immunoblotting enables precise quantification of membrane expression

  • Flow cytometry with extracellular epitope-targeted antibodies allows high-throughput analysis

  • Recent studies of SLC12A3 variants (p.E240K and p.L892P) used these techniques to demonstrate that mutations can affect membrane localization despite comparable plasma membrane fluorescence intensity

• Trafficking pathway dissection:

  • Synchronized trafficking assays with temperature blocks

  • Endosomal compartment co-localization studies using specific markers

  • Pulse-chase approaches with antibodies against extracellular epitopes

• Therapeutic screening applications:

  • High-content screening with SLC12A3 antibodies to identify compounds that rescue trafficking defects

  • Quantitative image analysis algorithms for automated phenotyping

  • Correlation of surface expression with functional sodium transport assays

These approaches are particularly valuable when investigating how disease-causing mutations like those found in Gitelman Syndrome affect SLC12A3 trafficking and function, potentially leading to therapeutic strategies for trafficking disorders .