SLC12A9 Antibody

What is SLC12A9 and why is it important in cellular physiology?

SLC12A9 is a member of the cation-chloride cotransporter (CCC) family that functions as a lysosomal ammonium exporter critical for preserving lysosomal homeostasis. It plays a fundamental role in detoxifying lysosomes by exporting ammonium (NH4+) in a chloride-dependent manner, preventing ammonium accumulation that would otherwise compromise lysosomal function . This function is particularly important because ammonia is a ubiquitous, toxic by-product of cell metabolism that can accumulate in its protonated form (ammonium) inside acidic lysosomes due to its high membrane permeability and proton affinity . SLC12A9 knockout cells exhibit grossly enlarged lysosomes and elevated ammonium content, demonstrating its essential role in maintaining lysosomal integrity .

How is SLC12A9 localized within cells?

SLC12A9 primarily localizes to lysosomal membranes within cells. When expressed as a fluorescently tagged protein (SLC12A9-GFP), it appears as punctate structures both in the perinuclear region and dispersed throughout the cytosol, indicating its presence on vesicular endomembranes rather than the plasma membrane . Co-localization studies have demonstrated high overlap between SLC12A9-GFP and endo-lysosomal markers LAMP1 and LAMP2, but not with markers of the trans-Golgi network (SYNT6) or early endosomes (RAB5) . This lysosomal localization is directed by two conserved targeting motifs: a dileucine-based motif (EXXXLL) and a tyrosine-based motif with a hydrophobic residue downstream (YXXL) .

What antibodies are available for SLC12A9 detection and what applications are they validated for?

Multiple antibodies are available for SLC12A9 detection from various providers. Based on the available data, some top validated antibodies include:

There are approximately 33 antibodies available from 9 different providers in total, with varying levels of validation .

How does SLC12A9 function as an ammonium-chloride co-transporter and what experimental approaches can verify this activity?

SLC12A9 functions as a chloride-driven ammonium co-transporter. Experimental evidence suggests that SLC12A9 requires chloride binding for ammonium transport, as mutations in conserved chloride-binding residues (Y313A and Y429A) impair its ability to rescue ammonium-induced phenotypes in SLC12A9 knockout cells . To verify SLC12A9's ammonium transport activity, researchers employed a strategic approach by leveraging a triple mutant SLC12A9 (LLY8/9/11/AAA) that mislocalized to the plasma membrane, allowing manipulation of the extracellular medium to test direct NH4+ import .

When exposed to 5 mM NH4+ in acidic conditions (pH 4.5 to mimic lysosomal pH), cells expressing the plasma membrane-localized SLC12A9 showed approximately 40% higher NH3+NH4+ content compared to control cells, providing evidence that SLC12A9 can transport NH4+ across biological membranes . To verify chloride dependence, researchers can generate point mutations in conserved chloride-binding residues (such as Y313A and Y429A) and assess whether these mutations abolish ammonium transport function .

What is the clinical significance of SLC12A9 expression in colorectal cancer and how can antibodies help evaluate this?

SLC12A9 shows significant upregulation in colorectal cancer (CRC) compared to normal tissue across multiple datasets, suggesting potential diagnostic and prognostic utility . Meta-analysis indicates that SLC12A9 has a standardized mean difference (SMD) of 1.42 (95% CI: 1.26-1.59) between CRC and normal tissue, with moderate diagnostic value (AUC = 0.78, 95% CI: 0.74-0.82) . Importantly, patients with overexpressed SLC12A9 demonstrate worse prognosis, and SLC12A9 expression correlates with several clinical characteristics including age, pathologic N stage, pathologic M stage, lymphatic invasion, and pathologic stage .

SLC12A9 antibodies can help evaluate this clinical significance through:

• Immunohistochemistry on patient tissue microarrays to assess expression levels correlated with clinical outcomes

• Western blotting to quantify protein expression in tumor versus adjacent normal tissue

• Flow cytometry to analyze SLC12A9 expression in circulating tumor cells or tumor-infiltrating immune cells

The diagnostic performance of SLC12A9 as a biomarker shows sensitivity of 0.92 (95% CI: 0.83-0.96) and specificity of 0.74 (95% CI: 0.69-0.78), suggesting potential clinical utility that can be further investigated using validated antibodies .

How does SLC12A9 expression correlate with immune cell infiltration in colorectal cancer, and what techniques can assess this relationship?

SLC12A9 expression in colorectal cancer demonstrates correlation with immune cell infiltration, which can be analyzed using computational methods like CIBERSORT applied to transcriptomic data . To experimentally investigate this relationship, researchers can employ:

• Multiplex immunohistochemistry using SLC12A9 antibodies alongside immune cell markers to visualize spatial relationships between SLC12A9-expressing cells and tumor-infiltrating immune cells

• Flow cytometry with validated SLC12A9 antibodies combined with immune cell markers to quantify proportions of different immune populations relative to SLC12A9 expression levels

• Single-cell RNA sequencing coupled with pseudo-time series analysis (as performed using monocle2 package) to examine developmental trajectories of immune cells in relation to SLC12A9 expression

Correlation analysis employing the Spearman method can identify specific immune cell populations that associate with SLC12A9 expression levels, potentially revealing mechanistic insights into how SLC12A9 might influence the tumor immune microenvironment .

What are the optimal protocols for immunohistochemical detection of SLC12A9 in tissue samples?

For optimal immunohistochemical detection of SLC12A9 in tissue samples, researchers should consider the following protocol guidelines:

• Tissue fixation and processing: Use 10% neutral-buffered formalin for fixation (24-48 hours), followed by paraffin embedding. Cut sections at 4-5 μm thickness.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective for SLC12A9 detection. Perform at 95-98°C for 20 minutes.

• Antibody selection:

  • For human tissue samples, polyclonal antibodies like Invitrogen's PA5-114359 or Novus Biologicals' NBP2-68858 have been validated for IHC applications .

  • For mouse tissues, consider species-specific antibodies with confirmed reactivity.

• Blocking and antibody incubation:

  • Block with 5% normal serum (matched to secondary antibody host) for 1 hour at room temperature

  • Incubate with primary antibody at optimized dilution (typically 1:100-1:500) overnight at 4°C

  • Use appropriate secondary antibody and detection system (HRP/DAB or fluorescent-based)

• Controls:

  • Include positive control tissues known to express SLC12A9 (e.g., macrophages express high levels of SLC12A9 relative to most other cell types)

  • Include negative controls (primary antibody omission)

  • Consider using SLC12A9 knockout tissues or cells as definitive negative controls when available

• Counterstaining: Hematoxylin for brightfield or DAPI for fluorescence microscopy

• Evaluation: Assess both intensity and proportion of SLC12A9 staining, particularly focusing on lysosomal localization patterns.

What are the key considerations when using SLC12A9 antibodies for studying its role in lysosomal function?

When investigating SLC12A9's role in lysosomal function using antibodies, researchers should consider:

• Subcellular localization verification:

  • Perform co-localization studies with established lysosomal markers (LAMP1, LAMP2) to confirm proper localization

  • Use confocal microscopy to accurately assess co-localization patterns

  • Consider super-resolution microscopy for detailed subcellular localization

• Functional validation approaches:

  • Complement antibody-based detection with functional assays measuring lysosomal pH (using LysoSensor probes) or ammonium content

  • Consider lysosomotropic dyes to assess lysosomal volume and morphology in relation to SLC12A9 expression

  • Use small fluorescent dextran (10 kDa) labeling to confirm lysosomal identity of compartments

• Experimental manipulations:

  • Assess antibody detection of SLC12A9 under conditions that alter lysosomal pH or ammonium levels

  • Consider chloride manipulation experiments to evaluate chloride-dependent functions

  • Compare antibody detection in wildtype versus SLC12A9 knockout/knockdown cells

• Technical considerations:

  • Optimize fixation methods to preserve lysosomal integrity (mild fixation often preferred)

  • Select detergents carefully for immunostaining or Western blot applications to maintain membrane protein conformation

  • Consider native vs. denaturing conditions for immunoprecipitation applications

• Mutant protein detection:

  • Verify antibody epitope locations relative to functional domains, particularly when studying mutant forms (Y313A and Y429A) that affect chloride binding

How should researchers validate SLC12A9 antibody specificity and functionality?

Thorough validation of SLC12A9 antibody specificity and functionality should include:

• Genetic validation:

  • Test antibody reactivity in SLC12A9 knockout/knockdown cells or tissues (gold standard for specificity)

  • Use siRNA/shRNA-mediated knockdown to demonstrate proportional reduction in antibody signal

  • Consider rescue experiments with wild-type SLC12A9 expression in knockout backgrounds

• Recombinant protein controls:

  • Test antibody reactivity against purified recombinant SLC12A9 protein

  • Compare detection of tagged recombinant SLC12A9 using both tag-specific antibodies and SLC12A9 antibodies

• Application-specific validation:

  • For Western blot: Verify single band of expected molecular weight (~75-80 kDa)

  • For IHC/ICC: Confirm expected subcellular pattern (punctate, lysosomal localization)

  • For flow cytometry: Establish appropriate gating strategies using positive and negative controls

• Cross-reactivity assessment:

  • Test reactivity against other SLC12 family members, particularly close homologs

  • Evaluate species cross-reactivity if working with multiple model organisms

• Epitope mapping:

  • When possible, determine the exact epitope recognized by the antibody

  • Assess whether mutations in key functional residues (Y313A, Y429A) affect antibody binding

• Lot-to-lot consistency:

  • Validate new lots against previously validated antibody preparations

  • Maintain consistent positive controls across experiments

How should researchers interpret changes in SLC12A9 expression in disease contexts like colorectal cancer?

When interpreting changes in SLC12A9 expression in disease contexts such as colorectal cancer, researchers should consider:

• Expression level quantification:

  • Use quantitative methods (qPCR, Western blot, digital pathology for IHC) rather than qualitative assessments

  • Normalize expression to appropriate housekeeping genes/proteins

  • Consider receiver operating characteristic (ROC) curve analysis to determine optimal cutoff values for "high" versus "low" expression

• Clinical correlation framework:

  • Assess SLC12A9 expression in relation to specific clinical characteristics (age, pathologic stage, lymphatic invasion)

  • Perform multivariate analyses to determine if SLC12A9 is an independent prognostic factor

  • Consider developing a nomogram incorporating SLC12A9 expression with other clinical features to predict survival outcomes

• Functional interpretation:

  • Connect expression changes to potential altered lysosomal function in cancer cells

  • Consider if SLC12A9 upregulation represents a compensatory response to increased metabolic ammonia production in cancer

  • Evaluate potential implications for therapy resistance mechanisms

• Contextual interpretation:

  • Assess SLC12A9 expression in relation to tumor microenvironment features

  • Consider cell type-specific expression (e.g., higher expression in macrophages)

  • Evaluate expression in different tumor regions (core, invasive front, metastases)

• Diagnostic and prognostic utility assessment:

  • Calculate sensitivity, specificity, and AUC values to evaluate diagnostic potential

  • Use Kaplan-Meier analysis with appropriate statistical testing to assess prognostic value

  • Compare the diagnostic/prognostic performance of SLC12A9 to established biomarkers

What experimental approaches can help resolve contradictory findings regarding SLC12A9 function or expression patterns?

When faced with contradictory findings regarding SLC12A9 function or expression patterns, researchers should implement the following approaches:

• Reconcile methodology differences:

  • Compare antibody clones, epitopes, and validation status across studies

  • Assess differences in experimental conditions (fixation methods, antigen retrieval, detection systems)

  • Consider tissue or cell type-specific differences in SLC12A9 expression and function

• Genetic manipulation validation:

  • Deploy CRISPR-Cas9 knockout followed by rescue experiments with wild-type and mutant SLC12A9

  • Use inducible expression systems to assess dose-dependent effects

  • Employ multiple genetic approaches (siRNA, shRNA, CRISPR) to confirm findings

• Contextual dependency exploration:

  • Investigate whether SLC12A9 function varies under different metabolic conditions

  • Assess if pH, ammonium levels, or chloride concentrations affect experimental outcomes

  • Consider developmental stage or disease progression as sources of apparent contradictions

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and functional assays to triangulate findings

  • Use single-cell approaches to resolve potential cell type-specific differences

  • Apply pseudo-time series analysis to understand temporal dynamics

• Reproducibility assessment:

  • Implement blinded analysis protocols

  • Increase sample sizes and perform power calculations

  • Pre-register key experiments and analysis plans

• Mechanistic dissection:

  • Focus on structure-function relationships using point mutations (e.g., Y313A and Y429A for chloride binding)

  • Investigate protein-protein interactions that might modify SLC12A9 function

  • Consider post-translational modifications that could explain functional differences

How can researchers accurately quantify SLC12A9 protein levels in complex biological samples?

Accurate quantification of SLC12A9 protein levels in complex biological samples requires careful methodological considerations:

• Western blot quantification:

  • Use validated antibodies with confirmed specificity

  • Include recombinant SLC12A9 protein standards at known concentrations

  • Implement standard curve calibration for absolute quantification

  • Use appropriate normalization controls (housekeeping proteins or total protein staining)

  • Employ digital image acquisition and analysis software with linear dynamic range

• Mass spectrometry approaches:

  • Develop targeted proteomics assays (PRM, MRM) for SLC12A9-specific peptides

  • Use stable isotope-labeled peptide standards for absolute quantification

  • Apply appropriate sample preparation techniques for membrane proteins

  • Consider enrichment strategies (immunoprecipitation) prior to MS analysis

• Immunohistochemistry quantification:

  • Implement digital pathology approaches with automated scoring algorithms

  • Use H-score or Allred scoring systems incorporating both intensity and proportion

  • Include calibration standards on each slide for normalization

  • Conduct inter-observer and intra-observer variability assessments

• Flow cytometry quantification:

  • Use antibody-binding capacity (ABC) beads to standardize measurements

  • Apply quantum calibration beads to convert fluorescence to molecules of equivalent soluble fluorochrome (MESF)

  • Consider intracellular versus surface staining protocols based on experimental question

  • Implement careful compensation when using multiple fluorophores

• ELISA/immunoassay development:

  • Generate standard curves using recombinant SLC12A9 protein

  • Validate assay specificity, sensitivity, precision, and accuracy

  • Determine matrix effects from complex biological samples

  • Consider sandwich ELISA format with capture and detection antibodies recognizing different epitopes

• Data normalization strategies:

  • For tissue samples: normalize to total protein, tissue area, or cell number

  • For cell culture: normalize to cell number, total protein, or appropriate reference genes

  • For fractionated samples: use compartment-specific markers (LAMP1/2 for lysosomal fraction)

What emerging techniques could advance our understanding of SLC12A9 biology and function?

Several emerging techniques show promise for advancing SLC12A9 research:

• Cryo-electron microscopy:

  • Determine the three-dimensional structure of SLC12A9 to understand its transport mechanism

  • Investigate structural changes during ammonium-chloride co-transport

  • Visualize interactions with other lysosomal proteins

• Live-cell imaging technologies:

  • Apply pH-sensitive fluorescent proteins fused to SLC12A9 to monitor activity in real-time

  • Use FRET-based sensors to detect conformational changes during transport

  • Implement lattice light-sheet microscopy for high-resolution 3D visualization of SLC12A9 dynamics

• CRISPR-based screening approaches:

  • Perform genome-wide CRISPR screens to identify genetic interactors of SLC12A9

  • Use CRISPRi/CRISPRa libraries to identify pathways affected by SLC12A9 expression modulation

  • Apply CRISPR base editing to introduce specific mutations without disrupting the entire gene

• Organoid and patient-derived models:

  • Evaluate SLC12A9 function in 3D organoid cultures from normal and cancer tissues

  • Generate patient-derived xenografts with modified SLC12A9 expression

  • Develop humanized mouse models to study SLC12A9 in an in vivo context

• Single-molecule imaging techniques:

  • Apply super-resolution microscopy (STORM, PALM) to visualize individual SLC12A9 molecules

  • Use single-molecule tracking to monitor SLC12A9 movement and clustering

  • Implement correlative light and electron microscopy (CLEM) to connect functional data with ultrastructural context

How might SLC12A9 antibodies contribute to therapeutic development for colorectal cancer?

SLC12A9 antibodies could facilitate therapeutic development for colorectal cancer through several avenues:

• Companion diagnostic development:

  • Use validated antibodies to develop IHC-based companion diagnostics

  • Identify patients likely to benefit from therapies targeting ammonium metabolism

  • Stratify patients based on SLC12A9 expression levels for clinical trials

• Therapeutic antibody development:

  • Generate antibodies targeting extracellular domains of SLC12A9

  • Develop antibody-drug conjugates for targeted delivery to SLC12A9-expressing cells

  • Create bispecific antibodies linking immune effectors to SLC12A9-expressing cancer cells

• Mechanism-based combination therapies:

  • Identify synergistic effects between SLC12A9 inhibition and standard therapies

  • Target metabolic vulnerabilities created by SLC12A9 overexpression

  • Develop rational combinations based on altered lysosomal function in SLC12A9-high tumors

• Immuno-oncology applications:

  • Investigate connections between SLC12A9 expression and immune cell infiltration patterns

  • Explore potential for combining SLC12A9-targeted therapies with immune checkpoint inhibitors

  • Assess impact of SLC12A9 modulation on tumor microenvironment

• Drug discovery platform development:

  • Establish high-throughput screening assays using SLC12A9 antibodies

  • Create cellular models with fluorescently tagged SLC12A9 for live-cell drug screening

  • Develop in silico modeling approaches based on antibody-derived structural information