SLC25A11 Antibody

What is SLC25A11 and why is it important in cellular metabolism?

SLC25A11 (Solute Carrier Family 25 Member 11) functions as the mitochondrial 2-oxoglutarate/malate carrier protein, catalyzing the transport of 2-oxoglutarate (alpha-oxoglutarate) across the inner mitochondrial membrane in an electroneutral exchange for malate. This protein is a critical component of the malate-aspartate shuttle (MAS), which is essential for maintaining redox balance by facilitating the transfer of reducing equivalents from cytosolic NADH to mitochondrial NADH for oxidative phosphorylation . Additionally, SLC25A11 contributes to several metabolic processes including the oxoglutarate/isocitrate shuttle, gluconeogenesis from lactate, and nitrogen metabolism, while also playing a role in maintaining mitochondrial morphology and organization of cristae .

What types of SLC25A11 antibodies are available for research applications?

Based on current research reagents, SLC25A11 antibodies are available in several formats:

Researchers should select antibodies based on their specific experimental needs, with options ranging from polyclonal antibodies (ABIN7235540) that recognize multiple epitopes to monoclonal antibodies that target specific amino acid sequences (e.g., AA 35-140, AA 50-200) .

How can I validate SLC25A11 antibody specificity for my experimental system?

Methodological approach for antibody validation:

• Perform western blotting with positive controls (e.g., Jurkat cells, HEK-293 cells, Raji cells, or human kidney tissue) which are known to express SLC25A11

• Include negative controls using SLC25A11 knockdown samples (via siRNA or shRNA)

• Verify the observed molecular weight matches the expected range (28-34 kDa)

• Conduct peptide competition assays to confirm binding specificity

• For cross-reactivity testing, compare antibody performance across human, mouse, and rat samples if working with multiple species

• Consider rescue experiments with SLC25A11 overexpression following knockdown to validate antibody specificity

What are the optimal conditions for using SLC25A11 antibodies in immunohistochemistry?

When designing IHC experiments with SLC25A11 antibodies, researchers should consider:

• Tissue preparation: Paraffin-embedded formalin-fixed tissues show successful staining results with SLC25A11 antibodies

• Antigen retrieval: Use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• Dilution optimization: Start with dilutions between 1:50-1:500 for IHC applications

• Positive control tissues: Human prostate tissue, human stomach cancer tissue, or lung cancer tissues which show reliable SLC25A11 expression

• Subcellular localization verification: Confirm cytoplasmic staining pattern (particularly for mitochondrial localization)

• Sample-dependent considerations: For cancer tissues, consider that SLC25A11 expression varies significantly between normal and cancerous tissue, being higher in NSCLC and melanoma but potentially lower in liver cancer

How do I design effective SLC25A11 knockdown experiments to study its function in cancer metabolism?

Based on published research methodologies:

• Select appropriate knockdown strategies:

  • siRNA-mediated transient knockdown shows 50-100% decrease in colony formation in cancer cell lines

  • shRNA-mediated stable knockdown provides long-term suppression for in vivo studies

  • CRISPR/Cas9 knockout systems are available for complete gene deletion

• Design time-course experiments:

  • Assess early metabolic effects at 24h (ATP production, OCR decrease)

  • Measure intermediate outcomes at 48h (40% cell death increase)

  • Evaluate late effects at 72h (up to 400% cell death increase)

• Include appropriate controls:

  • Normal cell lines such as IMR90 normal human lung fibroblasts which show no change in ATP production or proliferation upon SLC25A11 knockdown

  • Rescue experiments with SLC25A11 overexpression to confirm specificity

• Measure relevant metabolic parameters:

  • Mitochondrial membrane potential (decreases 30-40% in cancer cells)

  • Oxygen consumption rate (decreases 36-45% in cancer cells)

  • ATP production levels

  • Cell death via Annexin V staining

What are the recommended protocols for using SLC25A11 antibodies in Western blotting?

For optimal Western blotting results:

• Sample preparation:

  • Extract proteins from cells using RIPA buffer with protease inhibitors

  • For mitochondrial proteins, consider mitochondrial isolation protocols

  • Load 20-50 μg of total protein per lane

• Electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 28-34 kDa SLC25A11 protein

  • Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour

  • Incubate with primary SLC25A11 antibody at dilutions of 1:500-1:2000

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse depending on primary)

• Detection and controls:

  • Include positive control lysates (Jurkat cells, HEK-293 cells)

  • Use mitochondrial markers (e.g., VDAC, COX IV) as loading controls rather than standard housekeeping proteins

  • For subcellular localization studies, prepare cytosolic and mitochondrial fractions separately

How can SLC25A11 antibodies be utilized to study the role of the malate-aspartate shuttle in cancer metabolism?

Advanced methodological approaches:

• Combined immunofluorescence with metabolic flux analysis:

  • Use SLC25A11 antibodies for immunofluorescence localization (1:50-1:500 dilution)

  • Combine with seahorse XF analyzer measurements of oxygen consumption rates

  • Correlate SLC25A11 expression levels with oxygen consumption rates (shown to decrease by 36-45% upon knockdown)

• Co-immunoprecipitation studies:

  • Use SLC25A11 antibodies for immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg total protein)

  • Identify interacting partners within the malate-aspartate shuttle complex

  • Analyze the integrity of the shuttle complex in different metabolic states

• Metabolomic profiling in conjunction with antibody-based protein quantification:

  • Measure cytosolic and mitochondrial NADH levels

  • Quantify malate, glutamate, and aspartate concentrations

  • Correlate metabolite levels with SLC25A11 protein expression quantified by Western blotting

• In vivo imaging of shuttle activity:

  • Use fluorescent-labeled SLC25A11 antibodies for live cell imaging

  • Combine with NADH/NAD+ sensors to visualize shuttle activity

  • Study real-time changes in malate-aspartate shuttle during metabolic perturbations

How do mutations in SLC25A11 affect antibody binding and what are the implications for studying SLC25A11-related paraganglioma?

Advanced considerations when studying SLC25A11 mutations:

• Epitope mapping for mutation studies:

  • Select antibodies targeting regions distinct from known mutation hotspots

  • For paraganglioma research, use antibodies that recognize epitopes outside germline mutation sites

  • Consider using multiple antibodies targeting different regions (N-terminal, internal, C-terminal)

• Differential detection approaches:

  • Use wild-type specific and mutation-tolerant antibodies in parallel

  • For tumor-suppressor function studies, assess loss of heterozygosity using antibodies that can differentiate wild-type and mutant forms

• Functional correlation techniques:

  • Combine IHC with molecular profiling to identify "SDHx-like" molecular signatures in paraganglioma

  • Correlate antibody staining patterns with pseudohypoxic and hypermethylator phenotypes

  • Assess correlation between antibody detectability and functional assays of 2-oxoglutarate/malate transport

• Genotype-phenotype correlation studies:

  • Use antibody-based tissue microarrays to compare SLC25A11 expression between normal and tumor tissues with known mutation status

  • Correlate metastatic potential with specific mutations and antibody staining patterns

How do I interpret contradictory results when studying SLC25A11 expression in different cancer types?

Methodological approach to resolve contradictory findings:

• Tissue-specific expression patterns:

  • SLC25A11 is upregulated in NSCLC and melanoma tissues compared to normal controls

  • Conversely, SLC25A11 is downregulated in liver cancer compared to normal tissue

  • These differences may reflect tissue-specific metabolic adaptations rather than experimental inconsistencies

• Quantitative analysis strategies:

  • Use quantitative methods like qRT-PCR alongside antibody-based detection

  • Normalize expression data to appropriate tissue-specific reference genes

  • Consider both protein and mRNA levels when available to identify potential post-transcriptional regulation

• Correlation with clinical parameters:

  • In liver cancer, low SLC25A11 expression correlates with poor survival (shorter OS and RFS)

  • In NSCLC and melanoma, high SLC25A11 expression is associated with cancer development

  • Analyze clinical stage, histologic grade, and patient outcomes in relation to expression levels

• Functional context consideration:

  • SLC25A11 may have dual roles in cancer - supporting growth through ATP production while also regulating ROS via GSH transport

  • Different cancer types may prioritize different aspects of SLC25A11 function based on metabolic demands

  • Consider the broader metabolic context including glycolytic vs. oxidative phosphorylation dependence

What factors influence SLC25A11 antibody performance in experimental applications and how can I address them?

Critical factors and troubleshooting approaches:

• Epitope accessibility issues:

  • Problem: Mitochondrial localization may hinder antibody binding

  • Solution: Optimize permeabilization protocols with higher detergent concentrations for IF/IHC

  • Approach: Compare antibodies targeting different epitopes (e.g., AA 1-314, AA 35-140, AA 50-200)

• Post-translational modifications:

  • Problem: PTMs may mask epitopes or alter antibody recognition

  • Solution: Use dephosphorylation treatments prior to antibody application if phosphorylation is suspected

  • Approach: Select antibodies raised against recombinant full-length protein rather than synthetic peptides

• Isoform specificity:

  • Problem: Potential cross-reactivity with related mitochondrial carriers

  • Solution: Validate using knockout controls or competing peptides

  • Approach: Select antibodies validated against SLC25A11 knockout samples

• Fixation artifacts:

  • Problem: Formalin fixation may modify epitopes

  • Solution: Test alternative antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Approach: Compare fresh-frozen and FFPE samples when possible

• Technical variations in immunohistochemistry:

  • Problem: Inconsistent staining between experiments

  • Solution: Standardize protocols with positive control tissues (human prostate, stomach cancer)

  • Approach: Use automated staining platforms when available to reduce variability

How can SLC25A11 antibodies be utilized to explore the relationship between mitochondrial function and immune cell infiltration in cancer?

Innovative methodological approaches:

• Multiplex immunofluorescence techniques:

  • Co-stain tumor sections with SLC25A11 antibodies and immune cell markers (CD8, CD4, macrophages)

  • Analyze spatial relationships between SLC25A11-expressing cells and infiltrating immune cells

  • Quantify correlation patterns as observed in pancreatic cancer, where SLC25A11 shows positive correlation with CD8+ T cells (Partial. Cor = 0.154), CD4+ T cells (Partial. Cor = 0.314), and macrophages (Partial. Cor = 0.362)

• Single-cell analysis approaches:

  • Perform single-cell sorting based on SLC25A11 expression levels

  • Profile metabolic characteristics and cytokine production in isolated cells

  • Correlate SLC25A11 levels with immune cell functionality in the tumor microenvironment

• In vitro co-culture systems:

  • Establish co-cultures of cancer cells and immune cells

  • Manipulate SLC25A11 expression using CRISPR/Cas9 technology

  • Monitor immune cell migration, activation, and effector functions in relation to cancer cell SLC25A11 levels

• Metabolic interference studies:

  • Use SLC25A11 antibodies to track protein expression changes during metabolic perturbations

  • Assess how metabolic modulation affects both cancer cells and infiltrating immune cells

  • Develop therapeutic strategies targeting the metabolic interface between cancer and immune cells

What is the potential of SLC25A11 as a prognostic biomarker across different cancer types and how can antibody-based approaches advance this field?

Comprehensive biomarker development strategy:

• Multi-cancer tissue microarray analysis:

  • Perform large-scale IHC studies across cancer types using standardized SLC25A11 antibodies

  • Develop consistent scoring systems for expression levels

  • Compare prognostic value between cancer types where opposite trends have been observed:

    • Liver cancer: Low SLC25A11 associated with poor prognosis

    • NSCLC and melanoma: High SLC25A11 associated with cancer development

    • Paraganglioma: Germline mutations in SLC25A11 associated with metastatic disease

• Integrated multi-omics approach:

  • Combine antibody-based protein quantification with genomic and transcriptomic data

  • Correlate SLC25A11 protein levels with known mutations and expression patterns

  • Develop predictive models incorporating multiple data types for improved prognostic power

• Liquid biopsy development:

  • Investigate SLC25A11 protein detection in circulating tumor cells or exosomes

  • Develop sensitive immunoassays for detecting SLC25A11 in patient blood samples

  • Validate clinical utility through prospective studies correlating with patient outcomes

• Therapeutic response prediction:

  • Study SLC25A11 expression before and after treatment

  • Assess whether expression patterns predict response to metabolic-targeting therapies

  • Develop companion diagnostic approaches using validated SLC25A11 antibodies