SLC13A2 Antibody

What is SLC13A2 and why is it significant for metabolic research?

SLC13A2, also known as Na+-dicarboxylate cotransporter 1 (NaDC1), functions as a critical transporter of TCA cycle intermediates, particularly citrate. Its significance in metabolic research stems from its role in hepatocyte metabolic remodeling and liver regeneration processes. Recent studies demonstrate that SLC13A2 promotes import of citrate into hepatocytes, which serves as a building block for ACLY-dependent acetyl-CoA formation and de novo synthesis of cholesterol . Additionally, SLC13A2 has been identified as a tumor suppressor in hepatocellular carcinoma (HCC), where it is typically downregulated . Understanding SLC13A2's function provides insights into metabolic regulation, liver regeneration mechanisms, and potential therapeutic approaches for liver diseases and cancer.

What types of SLC13A2 antibodies are available for research and how do they differ?

SLC13A2 antibodies are available in several formats with distinct characteristics:

• Host species: Primarily rabbit and mouse-derived antibodies, with rabbit polyclonal antibodies being common for detection across multiple species .

• Clonality: Both monoclonal (e.g., clone 45 MI) and polyclonal antibodies are available, with monoclonals offering higher specificity for particular epitopes while polyclonals provide robust detection across multiple epitopes .

• Reactivity: Antibodies vary in their species reactivity profiles, with options available for human, rat, mouse, pig, cow, guinea pig, dog, rabbit, and chicken samples .

• Targeted regions: Various antibodies target different amino acid regions of SLC13A2 (e.g., AA 146-220, AA 345-455, AA 360-409, middle region, or C-terminus), allowing researchers to select based on structural or functional domains of interest .

These variations enable researchers to select antibodies optimized for their specific experimental model, detection method, and research question.

What are the validated applications for SLC13A2 antibodies?

SLC13A2 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): For detecting and quantifying SLC13A2 protein expression levels in tissue or cell lysates. This application is particularly useful for confirming knockout or overexpression efficiency in genetic modification studies .

• Immunohistochemistry (IHC): For localizing SLC13A2 in tissue sections, including paraffin-embedded and frozen sections, enabling spatial distribution analysis in organs like liver .

• Immunocytochemistry (ICC): For examining SLC13A2 expression and localization at the cellular level in cultured cells .

• Immunofluorescence (IF): For co-localization studies with other proteins or cellular structures .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection in solution-based assays .

When selecting an antibody, researchers should verify its validation status for their specific application and model organism, as performance can vary significantly between applications .

How can I verify SLC13A2 antibody specificity for my particular experimental model?

Verifying antibody specificity is crucial for generating reliable data. For SLC13A2 antibodies, implement these rigorous validation strategies:

• Genetic validation: Use tissues/cells with SLC13A2 knockout or knockdown. Recent studies employed AAV-mediated transduction of sgRNA targeting SLC13A2 in Cas9^fl/fl knock-in mice to create liver-specific SLC13A2 knockout models . The complete absence of signal in these models provides compelling evidence of antibody specificity.

• Overexpression controls: Parallel analysis of samples with experimentally overexpressed SLC13A2 allows confirmation of the antibody's ability to detect increased expression. This approach was successfully used in recent liver regeneration studies .

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of the immunizing peptide before application to your samples. Signal reduction or elimination indicates specificity for the target epitope.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of SLC13A2 to confirm consistent localization or expression patterns.

• Species-specific validation: When working with non-human models, verify that your chosen antibody has demonstrated reactivity in your species of interest through literature review or preliminary testing .

Each validation method strengthens confidence in antibody specificity, with genetic approaches providing the most definitive evidence.

How should I design experiments to investigate SLC13A2's role in metabolic regulation using antibody-based techniques?

Designing experiments to investigate SLC13A2's metabolic functions requires a multi-faceted approach:

• Expression correlation studies: Combine SLC13A2 immunodetection with metabolic phenotyping to establish correlations between SLC13A2 expression levels and metabolic parameters. For example, correlate SLC13A2 protein levels detected by Western blotting with measurements of citrate transport, cholesterol synthesis, or mitochondrial function .

• Temporal dynamics analysis: During processes like liver regeneration, analyze SLC13A2 expression at multiple timepoints (e.g., 1, 3, 5, and 7 days post-hepatectomy) to capture dynamic changes. Recent research demonstrated that SLC13A2 decreases rapidly after partial hepatectomy and recovers along with liver mass restoration .

• Co-localization with metabolic enzymes: Use dual immunofluorescence to examine spatial relationships between SLC13A2 and enzymes involved in citrate metabolism (e.g., ATP-citrate lyase) or cholesterol synthesis (e.g., HMGCR) .

• Gain/loss-of-function models: Combine genetic manipulation of SLC13A2 with antibody-based detection of downstream metabolic targets. For instance, researchers found that SLC13A2 overexpression increases cleavage of SREBP2 and expression of cholesterol metabolism genes like LDLR and HMGCR .

• Metabolic flux analysis integration: Correlate antibody-detected protein levels with metabolic flux measurements to establish functional relationships. Studies showed that SLC13A2-imported citrate leads to increased acetyl-CoA production, which affects PKM2 acetylation and subsequent metabolic pathways .

This comprehensive approach allows mechanistic insights into SLC13A2's role in metabolic regulation beyond simple correlative observations.

What are the critical controls when using SLC13A2 antibodies in cancer research models?

When investigating SLC13A2's tumor-suppressive role using antibodies, these critical controls are essential:

• Normal adjacent tissue controls: Always include normal tissue adjacent to tumors to establish baseline expression. Recent HCC studies identified SLC13A2 downregulation in tumor compared to adjacent normal liver tissue .

• Cell line validation panel: Test SLC13A2 antibody reactivity across multiple cell lines with known SLC13A2 expression levels. Include both HCC and normal hepatocyte cell lines (e.g., AML12) to confirm differential expression patterns .

• Isotype controls: Include appropriate isotype controls matched to your primary antibody to identify non-specific binding, particularly important in tumor tissues which often exhibit altered protein expression and increased background.

• Subcellular fraction controls: When examining localization, include markers for relevant subcellular compartments (plasma membrane, mitochondria, cytosol) to accurately determine SLC13A2 distribution, as its function depends on proper membrane localization.

• Expression gradient controls: If examining SLC13A2 in relation to tumor progression, include samples representing different disease stages to establish expression trends along the progression continuum.

• Treatment response controls: When evaluating effects of metabolic modulators (e.g., HMGCR inhibitors like lovastatin), include both pre- and post-treatment samples to assess SLC13A2 expression changes in response to intervention .

These controls ensure that observations about SLC13A2 in cancer research reflect true biological phenomena rather than technical artifacts.

What are the optimal sample preparation methods for SLC13A2 detection in different applications?

Optimal sample preparation varies by application and tissue type:

For Western Blotting:

• Membrane protein extraction: As SLC13A2 is a membrane transporter, use extraction buffers containing 1% NP-40 or Triton X-100 to effectively solubilize membrane proteins.

• Reducing conditions: Always include reducing agents (DTT or β-mercaptoethanol) in sample buffer to break disulfide bonds.

• Heat denaturation: Limit heating to 37°C for 30 minutes rather than boiling to prevent membrane protein aggregation.

• Loading control selection: Use Na+/K+-ATPase as a membrane-specific loading control rather than cytosolic proteins like GAPDH.

For Immunohistochemistry:

• Fixation: Use 4% paraformaldehyde fixation for 24-48 hours for optimal epitope preservation.

• Antigen retrieval: Employ citrate buffer (pH 6.0) heat-induced epitope retrieval, as this has been validated for SLC13A2 detection in liver tissues .

• Section thickness: Prepare 4-5 μm sections for optimal antibody penetration.

• Blocking: Use 5% normal serum from the same species as the secondary antibody plus 1% BSA to minimize background.

For Immunofluorescence:

• Cell fixation: Use 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: Apply 0.1% Triton X-100 for 10 minutes for access to intracellular epitopes.

• Antibody dilution: Use PBS with 1% BSA to maintain antibody stability and reduce non-specific binding.

• Counterstaining: Include DAPI nuclear staining and phalloidin for F-actin to establish cellular context for SLC13A2 localization.

These optimized protocols have been successfully employed in recent research examining SLC13A2's role in liver regeneration and tumor suppression .

How can I quantitatively analyze SLC13A2 expression data from antibody-based experiments?

Quantitative analysis of SLC13A2 expression requires appropriate methods for each technique:

For Western Blot Quantification:

• Densitometric analysis: Use software like ImageJ to measure band intensity, normalizing SLC13A2 signal to an appropriate loading control (preferably another membrane protein).

• Multiple exposure times: Capture several exposure times to ensure linearity of signal response for accurate quantification.

• Technical replicates: Run samples in triplicate across multiple blots to account for transfer variability.

• Standard curve inclusion: Include a dilution series of a reference sample for relative quantification across multiple experiments.

For IHC Quantification:

• H-score calculation: Calculate H-scores (0-300) based on staining intensity (0-3) multiplied by percentage of positive cells, as used in recent hepatic SLC13A2 studies .

• Automated analysis: Employ digital pathology software with trained algorithms to recognize SLC13A2-positive cells and quantify staining intensity.

• Regional analysis: Quantify expression separately in different liver zones (periportal vs. pericentral) to capture zonal expression differences.

• Double-labeled quantification: When co-staining with proliferation markers like Ki67, use co-localization analysis to determine the percentage of SLC13A2-positive cells that are also proliferating .

For IF Quantification:

• Fluorescence intensity measurement: Measure mean fluorescence intensity within defined cellular compartments.

• Co-localization coefficient calculation: Calculate Pearson's or Mander's coefficients when examining co-localization with metabolic enzymes or membrane markers.

• Z-stack analysis: Perform quantification across multiple z-planes to capture the three-dimensional distribution of SLC13A2.

Statistical approaches should include multiple biological replicates (n≥3) and appropriate statistical tests based on data distribution (parametric or non-parametric) with correction for multiple comparisons when necessary.

What are common pitfalls in SLC13A2 antibody experiments and how can they be avoided?

Researchers should be aware of these common pitfalls when working with SLC13A2 antibodies:

• Cross-reactivity with other SLC13 family members: SLC13A2 shares sequence homology with other family members, particularly SLC13A5. Confirm antibody specificity by checking for cross-reactivity with recombinant SLC13A5 and other family members.

• Inconsistent fixation effects: Different fixatives can dramatically affect epitope recognition. If transitioning between fresh-frozen and FFPE tissues, validate antibody performance in both conditions before making cross-sample comparisons.

• Cell type heterogeneity in tissue samples: Liver contains multiple cell types beyond hepatocytes. When quantifying SLC13A2 in liver samples, consider using hepatocyte-specific markers (HNF4α) for co-staining to ensure you're examining the intended cell population .

• Dynamic regulation during disease states: SLC13A2 expression changes significantly during liver regeneration and cancer progression . Ensure sampling timepoints are consistent when making comparisons across experimental groups.

• Buffer composition effects: The choice of lysis buffer can significantly impact membrane protein extraction. Avoid RIPA buffer for SLC13A2 analysis as it may disrupt membrane protein structure; instead use milder NP-40 or digitonin-based buffers.

• Antibody degradation: Store antibodies according to manufacturer recommendations, typically at -20°C in small aliquots to avoid freeze-thaw cycles. For the lyophilized format, reconstitute in the recommended volume of distilled water containing 0.09% sodium azide .

• Inappropriate controls: Failure to include genetic controls (knockout/overexpression) can lead to misinterpretation of non-specific signals. Recent studies used AAV-mediated manipulation of SLC13A2 expression as definitive controls .

By anticipating these challenges, researchers can design more robust experiments and generate more reliable data on SLC13A2 biology.

How can SLC13A2 antibodies be used to investigate the transporter's role in liver regeneration?

SLC13A2 antibodies are powerful tools for investigating this transporter's role in liver regeneration through these strategic approaches:

• Temporal expression profiling: Use immunoblotting and IHC to track SLC13A2 protein levels during the phases of liver regeneration (initiation, progression, termination). Recent research demonstrated that SLC13A2 decreases rapidly after partial hepatectomy and recovers with liver mass restoration, suggesting dynamic regulation during regeneration .

• Co-expression analysis with regeneration markers: Perform dual immunostaining of SLC13A2 with proliferation markers (Ki67, PCNA) and cell cycle regulators to establish correlations between SLC13A2 expression and hepatocyte proliferative status. This approach revealed that SLC13A2 overexpression increases Ki67-positive hepatocytes during liver regeneration .

• Zonal expression mapping: Map SLC13A2 expression across hepatic lobular zones during regeneration using IHC to identify spatial patterns that correlate with regenerative capacity. This is particularly relevant as periportal and pericentral hepatocytes exhibit different regenerative responses.

• Signaling pathway integration: Combine SLC13A2 immunodetection with phospho-specific antibodies for regenerative signaling pathways (ERK, mTOR, GSK3β) to establish mechanistic links. Studies found that SLC13A2 affects the activation of these pathways during regeneration .

• Metabolic enzyme co-regulation: Examine co-expression of SLC13A2 with enzymes involved in cholesterol biosynthesis (SREBP2, LDLR, HMGCR) using multiplexed immunofluorescence, as SLC13A2 promotes cholesterol synthesis required for membrane formation during hepatocyte proliferation .

• Experimental intervention assessment: Use immunostaining to evaluate SLC13A2 expression changes in response to regeneration-modulating interventions, such as lovastatin treatment, which was shown to abolish SLC13A2-mediated liver regeneration .

These approaches can provide mechanistic insights into how SLC13A2-mediated citrate transport contributes to the metabolic remodeling required for successful liver regeneration.

What emerging techniques can be combined with SLC13A2 antibodies to advance our understanding of its metabolic functions?

Integrating emerging techniques with SLC13A2 antibody-based detection can provide deeper insights into its metabolic functions:

• Spatial transcriptomics with protein verification: Combine spatial transcriptomics data on SLC13A2 mRNA with antibody-based protein detection to identify potential post-transcriptional regulation. This approach can reveal discrepancies between transcript and protein levels across tissue regions.

• Metabolic flux analysis with protein quantification: Integrate ^13C-labeled metabolite tracing with quantitative immunoblotting of SLC13A2 to correlate transporter levels with actual metabolic flux through pathways involving citrate. Recent research used this approach to demonstrate that SLC13A2 increases citrate import for acetyl-CoA production .

• Proximity labeling proteomics: Apply BioID or APEX2 proximity labeling fused to SLC13A2 to identify its protein interaction network, followed by verification with co-immunoprecipitation and immunoblotting for key interactors.

• Mass spectrometry imaging with immunohistochemistry: Correlate SLC13A2 protein localization with spatial distribution of metabolites (citrate, acetyl-CoA, cholesterol) using sequential tissue sections analyzed by IHC and mass spectrometry imaging.

• Single-cell proteomics validation: Verify single-cell protein analysis data of SLC13A2 with traditional immunofluorescence to understand cellular heterogeneity in transporter expression and its relation to metabolic phenotypes.

• Live-cell metabolic imaging: Combine fluorescent citrate sensors with immunofluorescence detection of fixed cells to correlate SLC13A2 expression with actual citrate transport activity at the single-cell level.

• CRISPR-mediated endogenous tagging: Use CRISPR to add small epitope tags to endogenous SLC13A2, allowing detection with highly specific anti-tag antibodies while maintaining native expression levels and regulation.

These integrated approaches move beyond simple detection toward functional understanding of SLC13A2's role in metabolic regulation in both normal physiology and disease states.

How can SLC13A2 antibodies contribute to investigating its potential as a therapeutic target in liver diseases and cancer?

SLC13A2 antibodies can significantly advance therapeutic target validation through these strategic applications:

• Expression profiling across disease progression: Use immunohistochemistry with SLC13A2 antibodies to characterize expression patterns across stages of liver disease and hepatocellular carcinoma. Recent research identified SLC13A2 downregulation in HCC tissues compared to adjacent normal liver, suggesting its potential as a biomarker .

• Therapeutic response monitoring: Apply immunodetection methods to assess changes in SLC13A2 expression following treatment with potential therapeutic agents. Studies demonstrated that manipulating SLC13A2 expression affects tumor growth, supporting its role as a target .

• Target validation in patient-derived models: Employ SLC13A2 antibodies in patient-derived xenografts and organoids to verify that expression patterns match those in primary tumors, confirming the relevance of findings from model systems.

• Mechanism-of-action studies: Use antibodies to track SLC13A2-dependent changes in PKM2 acetylation and degradation, as research has shown that citrate transported by SLC13A2 promotes PKM2 acetylation, leading to protein degradation and tumor suppression .

• Companion diagnostic development: Develop standardized IHC protocols with SLC13A2 antibodies as potential companion diagnostics to identify patients likely to respond to therapies targeting citrate metabolism.

• Combination therapy rational design: Apply multiplexed immunofluorescence to examine co-expression of SLC13A2 with other therapeutic targets, such as ACLY, which was shown to be mechanistically linked to SLC13A2's effects on cholesterol synthesis and cell proliferation .

• Normal tissue expression mapping: Use antibody-based tissue microarrays to comprehensively map SLC13A2 expression across normal tissues to predict potential off-target effects of therapies targeting this transporter.

These applications can help translate the fundamental understanding of SLC13A2 biology into clinically relevant therapeutic approaches for liver diseases and cancer.