SLC25A12 Antibody

What is SLC25A12 and why is it important in research?

SLC25A12 (also known as AGC1 or ARALAR1) is a mitochondrial electrogenic aspartate/glutamate antiporter that functions primarily in the malate-aspartate shuttle. Its importance stems from its crucial role in:

• Facilitating the exchange between intramitochondrial aspartate and cytosolic glutamate

• Supporting oxidative phosphorylation and ATP production through NADH transport

• Contributing to the transfer of reducing equivalents from cytosol to mitochondrial matrix

• Playing a key role in neuronal development, particularly in myelination processes

SLC25A12 has gained significant research attention due to its association with autism spectrum disorders (ASDs) and a neurodevelopmental syndrome characterized by seizures, hypotonia, arrested psychomotor development, and global hypomyelination .

What are the expression patterns of SLC25A12 across different tissues?

SLC25A12 shows distinct tissue-specific expression patterns:

SLC25A12 is the main AGC isoform present in the adult brain and is predominantly expressed in neurons. It is also the only AGC isoform expressed in pancreatic islets and β-cells, where it influences glucose-induced activation of mitochondrial metabolism and insulin secretion .

How does SLC25A12 function at the molecular level?

SLC25A12 functions as:

• A mitochondrial carrier embedded in the inner mitochondrial membrane

• A protein with six transmembrane alpha helices with N- and C-termini on the cytosolic side

• A calcium-sensitive transporter with several Ca²⁺-binding EF-hand motifs located on its long, hydrophilic amino-terminus

• An electrogenic antiporter that favors the efflux of aspartate and entry of glutamate and proton within the mitochondria

The protein catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate, which is an important step in urea synthesis. SLC25A12 also mediates the uptake of L-cysteinesulfinate by mitochondria in exchange for L-glutamate and proton, and can exchange L-cysteinesulfinate with aspartate in their anionic form without proton translocation .

What are the optimal applications and dilutions for SLC25A12 antibody use in different experimental techniques?

Based on validated antibody performance data:

For IHC applications, antigen retrieval is essential, with TE buffer at pH 9.0 generally providing better results, although citrate buffer at pH 6.0 may be used as an alternative .

How can I validate the specificity of an SLC25A12 antibody for my research?

Comprehensive validation should include:

• Western blot analysis with predicted band verification: SLC25A12 antibodies typically detect a band at approximately 74-75 kDa (predicted size) or sometimes at 63 kDa (observed size in some antibodies).

• Knockout/knockdown controls: Use SLC25A12 knockout tissue/cells as a negative control. Knockdown approaches (such as siRNA) can also be used to verify specificity.

• Peptide competition assays: Pre-incubation with the SLC25A12 blocking peptide should eliminate or significantly reduce immunoreactivity in all applications.

• Cross-species reactivity assessment: Test the antibody against samples from multiple species if your research requires cross-species studies.

• Multiple tissue verification: Test antibody performance across tissues with known differential expression (e.g., strong in brain and heart, weaker in kidney, minimal in liver).

An effective validation approach used in multiple studies involves comparing staining patterns before and after pre-incubation with blocking peptides, particularly in rat hippocampus immunohistochemical analyses, where specific staining should be suppressed by the blocking peptide .

What are the key considerations for using SLC25A12 antibodies in brain tissue analysis?

When analyzing SLC25A12 in brain tissue:

• Fixation protocols: For optimal preservation of mitochondrial antigens, 4% paraformaldehyde fixation is recommended, with careful timing to prevent overfixation.

• Region-specific considerations: Different brain regions show variable SLC25A12 expression patterns:

  • Highest expression in neurons, particularly in the cerebral cortex

  • Notable expression in cerebellar Purkinje cells

  • Detectable in hippocampal formation

• Developmental timing: SLC25A12 expression changes during development, which is particularly important when studying developmental disorders.

• Co-staining strategies: Combine SLC25A12 antibodies with:

  • Neuronal markers (calbindin, NeuN) for neuronal expression analysis

  • Myelin markers (MBP) to study relationship with myelination

  • Mitochondrial markers to confirm subcellular localization

• Background reduction: Brain tissue often exhibits higher background staining, so appropriate blocking (5-10% normal serum from the same species as the secondary antibody) is essential .

How is SLC25A12 implicated in neurodevelopmental disorders and what antibody-based approaches can be used to study these mechanisms?

SLC25A12 has significant implications in neurodevelopmental disorders:

• Autism Spectrum Disorders (ASDs):

  • SNPs in SLC25A12 (rs2056202 and rs2292813) show association with ASDs

  • Post-mortem samples from individuals with ASDs show approximately 1.5-fold higher SLC25A12 expression in the dorsolateral frontal cortex

  • Increased AGC1 activity reported in post-mortem samples from ASD patients

• Neurodevelopmental syndrome with global hypomyelination:

  • Homozygous mutations in SLC25A12 linked to severe phenotype with seizures, hypotonia, and arrested psychomotor development

Research approaches to investigate these connections include:

• Immunohistochemical analyses of post-mortem brain tissue to quantify expression levels in different brain regions

• Western blot quantification of protein levels in disease models

• Co-immunoprecipitation studies to identify altered interactions with other proteins

• Immunofluorescence co-localization studies to assess mitochondrial dysfunction in neurons

These antibody-based approaches have revealed that SLC25A12 dysfunction affects myelination processes and neuronal structure, which may contribute to the pathogenesis of these disorders .

What research models are available for studying SLC25A12 function, and how are antibodies employed in these models?

Key research models include:

• Slc25a12-knockout mice:

  • Born normally but show delayed development and die around 3 weeks after birth

  • Display smaller brains with reduction in myelin basic protein (MBP)-positive fibers

  • Exhibit abnormal neurofilamentous accumulations in neurons

  • Show Purkinje cell abnormalities in the cerebellum

• Brain slice cultures:

  • Cerebellar slice cultures from knockout mice demonstrate myelination defects

  • Myelin deficits can be reversed by pyruvate administration

• Primary oligodendrocyte cultures:

  • Reduction of Slc25a12 in rat primary oligodendrocytes leads to cell-autonomous reduction in MBP expression

Antibody applications in these models include:

• Validation of knockout status via immunoblotting

• Tracking developmental expression changes with immunohistochemistry

• Quantifying myelination defects using anti-MBP staining

• Assessing neuronal integrity with neurofilament and calbindin antibodies

• Monitoring AGC1 expression in response to therapeutic interventions

These approaches have been instrumental in establishing that AGC1 activity influences myelination and neuronal structure, with implications for neurodevelopmental disorders .

What is the relationship between SLC25A12 expression and cancer, and how can antibodies help investigate this connection?

The relationship between SLC25A12 and cancer is complex:

• Prognostic implications:

  • SLC25A12 expression is positively correlated with worse patient prognosis in several cancer types

  • This correlation is particularly strong in Cervical Squamous Cell Carcinoma (CESC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), and Sarcoma (SARC)

• Metastasis influence:

  • AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis

  • This suggests that certain branches of metabolism impact tumor growth and tumor metastasis differently

Research approaches using antibodies include:

• Immunohistochemical analysis of tumor tissues to assess SLC25A12 expression levels

  • Validated in human breast cancer, pancreatic cancer, and stomach cancer tissues

• Western blot quantification in cancer cell lines

  • Particularly useful in HeLa, A431, and Jurkat cell lines

• Tissue microarray analysis of patient samples for correlation with clinical outcomes

These studies highlight that while SLC25A12 may be necessary for optimal tumor growth, its influence on metastatic potential appears to operate through different mechanisms, making it a potentially important target for understanding cancer metabolism .

What are common issues in SLC25A12 antibody applications and how can they be resolved?

For mitochondrial proteins like SLC25A12, tissue preparation is critical. Fresh tissue is optimal, and particular attention should be paid to fixation protocols that preserve mitochondrial architecture without masking epitopes .

How can I optimize SLC25A12 antibody protocols for detecting subtle expression differences in disease models?

Optimizing detection of subtle expression differences requires:

• Quantitative Western blot approaches:

  • Use loading controls carefully selected for stability in your disease model

  • Consider fluorescent-labeled secondary antibodies for more precise quantification

  • Implement technical replicates (3-4 minimum) and biological replicates (different animals/patients)

  • Use gradient gels for better resolution of SLC25A12 isoforms

• Enhanced immunohistochemistry protocols:

  • Employ tyramide signal amplification for low abundance detection

  • Utilize confocal microscopy with z-stack analysis for precise localization

  • Consider RNAscope® combined with IHC for simultaneous mRNA and protein detection

  • Use stereological approaches for unbiased quantification

• Cell-type specific analysis:

  • Implement double or triple immunolabeling to identify cell-type specific changes

  • Use laser capture microdissection combined with Western blot for region-specific analysis

  • Consider flow cytometry with permeabilization for population-level quantification

These approaches have been successfully used to detect the 1.5-fold expression difference in SLC25A12 in post-mortem samples from individuals with ASDs compared to controls .

What emerging techniques are being developed for studying SLC25A12 in living systems?

Cutting-edge approaches for SLC25A12 research include:

• CRISPR-Cas9 gene editing:

  • Generation of cell lines with patient-specific mutations

  • Development of conditional knockout models for temporal control

  • Creation of fluorescent protein fusions at endogenous loci

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize mitochondrial localization

  • Live-cell imaging of SLC25A12 trafficking and dynamics

  • Multi-photon imaging in intact tissue with antibody-based probes

• Functional assays:

  • Real-time monitoring of aspartate/glutamate exchange activity

  • Mitochondrial respirometry combined with immunocytochemistry

  • Metabolic flux analysis in SLC25A12-manipulated systems

• Single-cell approaches:

  • Single-cell proteomics to detect cell-specific expression levels

  • Combined transcriptome and proteome analysis at the single-cell level

  • Spatial transcriptomics with protein confirmation via antibodies

These emerging techniques are expected to provide deeper insights into SLC25A12's role in normal development and disease states, potentially identifying new therapeutic targets for neurodevelopmental disorders .

How can SLC25A12 antibodies be utilized in therapeutic development and biomarker identification?

SLC25A12 antibodies offer several avenues for translational research:

• Therapeutic target validation:

  • Identifying specific cell populations where SLC25A12 modulation might be beneficial

  • Validating effects of small molecule modulators on SLC25A12 expression/function

  • Monitoring off-target effects of therapeutic interventions

• Biomarker development:

  • Assessing SLC25A12 levels in accessible tissues (blood cells, skin fibroblasts) as potential biomarkers for neurological disorders

  • Correlating SLC25A12 expression with disease progression in longitudinal studies

  • Evaluating post-translational modifications as disease-specific markers

• Patient stratification:

  • Using SLC25A12 antibodies to identify patient subgroups that might respond to metabolism-targeted therapies

  • Correlating expression patterns with genetic variants for personalized medicine approaches

• Drug screening platforms:

  • Developing high-content screening assays using SLC25A12 antibodies to identify compounds that normalize expression or function

  • Creating cell-based assays to monitor SLC25A12-dependent metabolic changes

The role of SLC25A12 in both neurodevelopmental disorders and cancer makes it a particularly interesting target for therapeutic development, with antibody-based approaches providing crucial tools for validation and monitoring .