SLC13A5 Antibody

What is SLC13A5 and why is it important for metabolic research?

SLC13A5 (Solute Carrier Family 13 Member 5), also known as NaCT (Sodium-coupled Citrate Transporter), is a high-affinity sodium/citrate cotransporter that mediates the electrogenic entry of citrate into cells. This transporter recognizes the trivalent form of citrate rather than the divalent form . SLC13A5 plays critical roles in multiple metabolic pathways where citrate is essential, including:

• Energy production via the Krebs cycle

• Fatty acid synthesis

• Cholesterol synthesis

• Glycolysis regulation

• Gluconeogenesis

SLC13A5 is particularly important in research because disruptions in its function are associated with developmental disorders and epilepsy, while its modulation offers potential therapeutic approaches for metabolic disorders .

For optimal detection of SLC13A5 in tissue samples:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours at room temperature.

• Process and embed in paraffin following standard protocols.

• Section tissues at 4-6 μm thickness.

• For antigen retrieval, use citrate buffer (pH 6.0) with heat-mediated protocols .

• For Western blotting of membrane proteins like SLC13A5:

  • Use specialized membrane protein extraction buffers containing mild detergents.

  • Avoid boiling samples to prevent protein aggregation.

  • Include protease inhibitors in all buffers to prevent degradation.

  • Consider using 12% SDS-polyacrylamide gels for optimal resolution .

How can researchers distinguish between different molecular phenotypes of SLC13A5 mutations using antibody-based techniques?

Recent studies have identified two distinct classes of SLC13A5 mutations with different molecular phenotypes that can be distinguished using antibody-based techniques :

Class I Mutations (e.g., C50R, T142M, T227M):

• Normal protein expression levels at the cell surface

• Impaired citrate transport function

• Normal glycosylation patterns

• Western blot: Normal band size and intensity

• Immunofluorescence: Normal membrane localization

Class II Mutations (e.g., G219R, S427L, L488P):

• Reduced protein expression

• ER retention

• Immature core-glycosylation

• Shortened half-lives

• Western blot: Reduced band intensity and/or altered migration pattern

• Immunofluorescence: Predominant ER localization

Methodological approach:

• Perform Western blotting with glycosylation-specific analyses (PNGase F or Endo H treatment)

• Conduct subcellular fractionation followed by immunoblotting

• Use dual-label immunofluorescence with ER markers (e.g., calnexin) and SLC13A5 antibodies

• Measure protein half-life using cycloheximide chase experiments

What are the recommended controls when studying SLC13A5 function using knockout/knockdown models?

When studying SLC13A5 function using knockout or knockdown approaches, implement these essential controls:

Positive Controls:

• Wild-type cells/tissues with confirmed SLC13A5 expression

• For citrate transport assays: cells overexpressing SLC13A5

Negative Controls:

• SLC13A5 knockout cells/tissues

• siRNA-NC (negative control) transfected cells

• Empty vector-transfected cells

• Sodium-free conditions during transport assays to demonstrate sodium-dependency

Validation Controls:

• qRT-PCR to confirm knockdown at mRNA level

• Western blot to confirm protein reduction

• Functional assays to measure citrate transport (using [14C]-citrate)

• Measurement of serum and CSF citrate levels (elevated citrate is a functional marker of SLC13A5 deficiency)

For in vivo models, researchers can reference the comprehensive collection of available SLC13A5 models in the literature, including global knockout mice, conditional knockout mice, and zebrafish models .

How can SLC13A5 antibodies be used to investigate the PXR-mediated regulation of SLC13A5 expression?

To investigate PXR-mediated regulation of SLC13A5 expression using antibody-based approaches:

• Cell Treatment Design:

  • Treat human primary hepatocytes with PXR activators (e.g., rifampicin at appropriate concentrations)

  • Include appropriate vehicle controls

  • Consider time-course experiments (24-72 hours)

• Protein Expression Analysis:

  • Perform Western blot analysis using anti-SLC13A5 antibodies (1:200 dilution)

  • Include β-actin (1:5000) as loading control

  • Quantify relative protein levels using densitometry

• Subcellular Localization Studies:

  • Conduct immunofluorescence or immunohistochemistry to determine if PXR activation affects SLC13A5 localization

  • Co-stain with membrane markers to confirm proper trafficking

• Chromatin Immunoprecipitation:

  • Use ChIP assays with anti-PXR antibodies to identify PXR binding to SLC13A5 enhancer regions

  • Focus on the two identified enhancer modules located at -22kb and -1.7kb upstream of the SLC13A5 transcription start site

  • Design primers for these regions: for example, 5′-CGGGCTAGCCTTCAGTCTCCACCCCAAGAT-3′ and 5′-ATGTACCCTGACTATGCCTTC-3′ for one module

• Luciferase Reporter Assays:

  • Use reporter constructs containing SLC13A5 promoter and enhancer elements (e.g., SLC13A5-1k/DR4-1/2)

  • Co-transfect with PXR expression vectors

  • Measure luciferase activity following PXR activator treatment

What are the critical factors for optimizing Western blot protocols for SLC13A5 detection?

Optimizing Western blot protocols for SLC13A5 detection requires attention to several critical factors:

• Sample Preparation:

  • Use 12% SDS-polyacrylamide gels for optimal resolution

  • Homogenize samples in buffer containing appropriate detergents for membrane protein extraction

  • Include protease inhibitors to prevent degradation

• Antibody Selection and Dilution:

  • Primary antibody: 1-5 μg/mL (monoclonal) or manufacturer-recommended dilution

  • Secondary antibody: HRP-conjugated at 1:5000-1:10,000 dilution

  • Consider using antibodies validated for Western blot applications

• Blocking Conditions:

  • 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20)

  • Block for 1 hour at room temperature to reduce background

• Detection Method:

  • Enhanced chemiluminescence (ECL) is recommended for optimal sensitivity

  • For quantification, use β-actin (1:5000) as a loading control

• Special Considerations:

  • As a membrane protein, SLC13A5 may form aggregates if boiled; heat samples at 37°C instead

  • For glycosylation analysis, include PNGase F or Endo H treatment controls

  • When studying mutant variants, longer exposure times may be needed for detection of poorly expressed proteins

How should researchers interpret contradictory citrate measurements in different tissue types from SLC13A5 knockout models?

When interpreting contradictory citrate measurements across different tissues in SLC13A5 knockout models, consider these methodological approaches:

• Understanding Tissue-Specific Differences:

  • In SLC13A5 knockout mice, citrate levels decrease in parahippocampal cortex but not in hippocampus

  • Citrate increases in osteoblasts, suggesting potential tissue-specific compensatory mechanisms

• Methodological Approach to Resolving Contradictions:

  • Measure citrate levels using multiple complementary techniques (LC-MS/MS, enzymatic assays)

  • Analyze both intracellular and extracellular (media, serum, CSF) citrate concentrations

  • Perform time-course experiments to capture dynamic changes

  • Investigate mitochondrial vs. cytosolic citrate pools separately

  • Examine expression of other citrate transporters that might compensate (e.g., other SLC13 family members)

• Species Differences Considerations:

  • Human SLC13A5 is primarily expressed in liver with lower brain expression, yet mutations cause severe neurological phenotypes

  • Human NaCT is a low-affinity, high-capacity transporter highly selective for citrate

  • Rodent NaCT transports citrate and succinate equally well

  • Consider using humanized mouse models for more translatable results

• Experimental Controls:

  • Include both wild-type controls and heterozygous animals

  • Measure other TCA cycle intermediates to assess metabolic compensation

  • Examine the impact of dietary citrate supplementation

What methodological approaches should be used when validating novel SLC13A5 antibodies?

A comprehensive validation approach for novel SLC13A5 antibodies should include:

• Specificity Testing:

  • Western blot comparison using:

    • Wild-type samples with known SLC13A5 expression

    • SLC13A5 knockout tissues/cells as negative controls

    • SLC13A5 overexpression systems as positive controls

  • Peptide competition assays to confirm epitope specificity

  • Cross-reactivity testing against other SLC family members

  • siRNA knockdown validation (e.g., using siRNA-SLC13A5 at 100nM concentration)

• Applications Validation:

  • Western blotting with titration series (0.1-10 μg/mL)

  • Immunohistochemistry on formalin-fixed, paraffin-embedded tissues

  • Immunofluorescence in relevant cell lines (e.g., HepG2, primary hepatocytes)

  • Immunoprecipitation followed by mass spectrometry confirmation

  • ELISA development and sensitivity testing

• Reproducibility Assessment:

  • Batch-to-batch consistency evaluation

  • Inter-laboratory validation

  • Testing across multiple lots of the same antibody

• Species Cross-Reactivity:

  • Testing against human, mouse, and rat samples based on epitope conservation

  • Sequence alignment analysis to predict cross-reactivity

  • Experimental validation in multiple species

• Documentation and Reporting:

  • Detailed protocols including all critical parameters

  • Complete description of validation methods and results

  • Publication of validation data following antibody reporting guidelines

How can researchers address challenges in detecting low-abundance SLC13A5 in neuronal tissues?

Detection of low-abundance SLC13A5 in neuronal tissues presents unique challenges. Address these with the following methodological approaches:

• Sample Enrichment Strategies:

  • Perform subcellular fractionation to concentrate membrane proteins

  • Use immunoprecipitation to enrich SLC13A5 before Western blot analysis

  • Consider proximity ligation assay (PLA) for increased sensitivity in tissue sections

• Signal Amplification Methods:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Use high-sensitivity ECL substrates for Western blotting

  • Consider using biotinylated secondary antibodies with streptavidin-HRP systems

• Optimized Antigen Retrieval:

  • For FFPE tissues, perform heat-mediated antigen retrieval with citrate buffer (pH 6.0)

  • Extend retrieval time to 20-30 minutes for difficult samples

  • Consider alternative retrieval buffers (EDTA, pH 8.0) if citrate buffer yields poor results

• Alternative Detection Approaches:

  • Use mRNA detection methods (RNAscope, in situ hybridization) to confirm expression patterns

  • Consider reporter mouse models (e.g., SLC13A5-GFP) for expression studies

  • Employ single-cell approaches to identify specific neuronal populations expressing SLC13A5

• Controls and Validation:

  • Include positive control tissues with known high expression (e.g., liver)

  • Use patient-derived cells with SLC13A5 mutations as comparative controls

  • Consider parallel detection with multiple antibodies recognizing different epitopes

What are the optimal methods for analyzing SLC13A5 glycosylation status when studying disease-causing mutations?

For analyzing SLC13A5 glycosylation status in the context of disease-causing mutations:

• Glycosidase Treatment Analysis:

  • Treat protein samples with PNGase F (removes all N-linked glycans)

  • Treat parallel samples with Endo H (removes only high-mannose, immature glycans)

  • Compare migration patterns by Western blot:

    • Endo H-resistant bands indicate mature, complex glycosylation (post-Golgi)

    • Endo H-sensitive bands suggest immature glycosylation (ER retention)

• Lectin Blotting:

  • Use specific lectins to distinguish glycan types:

    • Concanavalin A (Con A) for high-mannose structures

    • Wheat germ agglutinin (WGA) for complex N-glycans

  • Perform parallel Western blots with SLC13A5 antibodies and lectins

• Pulse-Chase Experiments:

  • Label newly synthesized proteins with radioactive amino acids or click chemistry approaches

  • Chase for various time periods (0-24h)

  • Immunoprecipitate SLC13A5

  • Analyze glycosylation status changes over time

• Co-localization Studies:

  • Perform dual immunofluorescence with:

    • Anti-SLC13A5 antibodies

    • Markers for ER (calnexin, calreticulin)

    • Markers for Golgi (GM130, TGN46)

  • Quantify co-localization coefficients between SLC13A5 and organelle markers

• Mass Spectrometry Analysis:

  • Immunoprecipitate SLC13A5 from cells expressing wild-type or mutant proteins

  • Perform glycopeptide analysis by mass spectrometry

  • Identify specific glycosylation sites and glycan structures

This approach allows classification of SLC13A5 mutations into distinct molecular phenotypes based on their effects on glycosylation and protein trafficking .

How can researchers effectively use SLC13A5 antibodies in patient-derived iPSC models for studying pathogenic mechanisms?

For effective use of SLC13A5 antibodies in patient-derived iPSC models:

• iPSC Model Selection and Validation:

  • Utilize available patient-derived iPSC lines with specific SLC13A5 mutations

  • Include isogenic corrected controls when possible

  • Confirm pluripotency markers and genomic integrity before differentiation

• Differentiation Protocols:

  • For neuronal studies: differentiate iPSCs into neural precursor cells (NPCs) and mature neurons

  • For metabolic studies: differentiate into hepatocyte-like cells

  • Consider organoid models for 3D tissue-like structures

• SLC13A5 Expression Analysis:

  • Monitor expression during differentiation using Western blot

  • Perform immunofluorescence to assess subcellular localization

  • Use quantitative approaches (flow cytometry, high-content imaging) for comparative analysis

• Functional Assessments with Antibody-Based Methods:

  • Correlate protein expression with citrate transport using [14C]-citrate uptake assays

  • Assess co-localization with organelle markers to determine trafficking defects

  • Use proximity ligation assays to study protein-protein interactions

• Disease Mechanism Studies:

  • Analyze metabolic consequences using metabolomics in combination with SLC13A5 protein quantification

  • Study mitochondrial function in relation to SLC13A5 expression levels

  • Investigate effects on neuronal excitability in iPSC-derived neurons

• Rescue Experiments:

  • Test therapeutic approaches (small molecules, gene therapy) and monitor SLC13A5 expression/localization

  • For Class II mutations: test whether protein folding correctors restore SLC13A5 trafficking

  • For Class I mutations: evaluate whether transport enhancers improve function

How can SLC13A5 antibodies contribute to understanding the relationship between citrate transport and epileptogenesis?

SLC13A5 antibodies offer several methodological approaches to investigate the relationship between citrate transport and epileptogenesis:

• Expression Analysis in Epilepsy Models:

  • Compare SLC13A5 expression and localization in:

    • Animal models of epilepsy (e.g., kainic acid, pilocarpine models)

    • Human epileptic brain tissue versus controls

    • Developmental time points (critical for understanding early-onset seizures in SLC13A5 deficiency)

• Cell-Type Specific Studies:

  • Perform co-immunostaining with neuronal, astrocytic, and microglial markers

  • Quantify SLC13A5 expression in specific neuronal subtypes (excitatory vs. inhibitory)

  • Correlate with electrophysiological properties

• Mechanistic Investigations:

  • Study effects of altered SLC13A5 expression on:

    • Mitochondrial function and energy metabolism

    • Neurotransmitter synthesis and recycling

    • Membrane excitability and ion channel function

• Therapeutic Target Validation:

  • Monitor SLC13A5 expression/localization after treatment with antiseizure medications

  • Test whether citrate supplementation affects SLC13A5 expression or localization

  • Evaluate if metabolic interventions (ketogenic diet) alter SLC13A5 distribution or function

• Integration with Metabolomics:

  • Correlate tissue-specific SLC13A5 protein levels with citrate concentrations and other metabolites

  • Investigate citrate's role as a chelator of calcium, potentially affecting neuronal excitability

  • Study relationships between SLC13A5, citrate transport, and energy metabolism in epileptic brain tissue

What approaches can be used to investigate potential compensation mechanisms in SLC13A5 deficiency models?

To investigate compensation mechanisms in SLC13A5 deficiency models:

• Comprehensive Transporter Expression Profiling:

  • Perform Western blot analysis of other SLC family transporters in SLC13A5-deficient models

  • Focus on:

    • Other TCA cycle transporter families (SLC25)

    • Monocarboxylate transporters (SLC16)

    • Other sodium-coupled transporters

• Pathway Analysis:

  • Examine mitochondrial citrate production enzymes (aconitase, citrate synthase) by Western blot

  • Investigate fatty acid metabolism enzymes (ATP citrate lyase, acetyl-CoA carboxylase)

  • Study gluconeogenesis pathway components

• Temporal Compensation Analysis:

  • Track expression changes over developmental stages or disease progression

  • Compare acute versus chronic models of SLC13A5 deficiency

  • Analyze early compensatory responses versus long-term adaptations

• Functional Complementation Studies:

  • Systematically overexpress candidate compensatory transporters in SLC13A5-deficient models

  • Measure rescue of phenotypes (citrate transport, lipid accumulation)

  • Use combinatorial knockdown approaches to identify synthetic interactions

• Tissue-Specific Compensation Analysis:

  • Compare compensation mechanisms between tissues with different phenotypic outcomes:

    • Parahippocampal cortex (decreased citrate) versus hippocampus (normal citrate)

    • Osteoblasts (increased citrate) versus other cell types

  • Consider species-specific differences in compensatory mechanisms

How can researchers use antibody-based methods to explore the relationship between SLC13A5 function and lipid metabolism?

To investigate SLC13A5's role in lipid metabolism using antibody-based approaches:

• Lipid Droplet Analysis in Relation to SLC13A5 Expression:

  • Perform siRNA knockdown of SLC13A5 (100 nM) in HepG2 cells

  • Stain lipid droplets with BODIPY and quantify using ImageJ software

  • Correlate lipid content with SLC13A5 protein levels by Western blot

  • Treat cells with rifampicin to enhance lipid accumulation through PXR-mediated SLC13A5 induction

• Co-localization Studies:

  • Perform dual immunofluorescence with:

    • Anti-SLC13A5 antibodies

    • Lipid droplet markers (PLIN2/ADRP, PLIN3/TIP47)

    • Fatty acid synthesis enzymes (FASN, ACC)

  • Quantify spatial relationships using confocal microscopy

• Protein-Protein Interaction Analysis:

  • Conduct co-immunoprecipitation with SLC13A5 antibodies

  • Probe for interactions with:

    • ATP citrate lyase (converts citrate to acetyl-CoA for lipid synthesis)

    • Acetyl-CoA carboxylase (rate-limiting enzyme in fatty acid synthesis)

    • PXR (transcriptional regulator of SLC13A5)

• Pathway Activation Studies:

  • Monitor effects of SLC13A5 modulation on lipogenic pathways:

    • SREBP-1c activation and nuclear translocation

    • ChREBP phosphorylation status

    • PPARγ expression and activity

  • Use Western blotting with pathway-specific antibodies

• Clinical Correlation Studies:

  • Analyze SLC13A5 expression in patient liver samples

  • Correlate with steatosis grade and lipid accumulation markers

  • Investigate associations with metabolic syndrome parameters

  • Study effects of SLC13A5 variants on lipid profiles in patient cohorts