SLC25A37 Antibody

What is SLC25A37 and why is it important in research?

SLC25A37 (Solute Carrier Family 25 Member 37), also known as Mitoferrin-1 (MFRN1), functions as a mitochondrial iron transporter that specifically mediates iron uptake in developing erythroid cells. This protein plays an essential role in heme biosynthesis by delivering iron, presumably as Fe(2+), into the mitochondria where it's incorporated into protoporphyrin IX to make heme . Recent research has also identified SLC25A37 as an organ-specific requirement for cancer metastasis, particularly in liver metastasis models . Its critical role in iron homeostasis makes it an important research target for understanding both normal physiological processes and disease mechanisms, particularly in hematological disorders and certain cancer types.

What applications are SLC25A37 antibodies suitable for?

SLC25A37 antibodies have been validated for multiple research applications, making them versatile tools for investigating this protein. Common applications include:

• Western Blotting (WB): For detecting and quantifying SLC25A37 protein in cell or tissue lysates

• Immunohistochemistry (IHC): For visualizing the protein in fixed tissue sections

• Flow Cytometry (FACS): For analyzing SLC25A37 expression in individual cells

• Immunofluorescence (IF/ICC): For determining subcellular localization in fixed cells

• ELISA: For quantitative detection of SLC25A37 in samples

When selecting an antibody for your research, verify that it has been validated for your specific application and species of interest, as reactivity can vary between products.

How do I choose the appropriate SLC25A37 antibody for my research?

When selecting a SLC25A37 antibody, consider several critical factors:

• Target epitope: Different antibodies target various regions of the protein. Some target the N-terminal region (AA 1-44), others the C-terminal region (AA 309-338), and some the middle section (AA 65-338) . Epitope selection may affect antibody performance depending on protein conformation or interactions in your experimental system.

• Species reactivity: Verify that the antibody recognizes SLC25A37 in your species of interest. Available antibodies have different reactivity profiles, with some recognizing only human SLC25A37, while others cross-react with rat and/or mouse orthologs .

• Application validation: Ensure the antibody has been validated for your specific application. Check if the manufacturer provides validation data for Western blot, IHC, IF, FACS, or ELISA as appropriate for your needs .

• Clonality: Most available SLC25A37 antibodies are polyclonal, generated in rabbits . Consider whether a polyclonal antibody's broader epitope recognition or a monoclonal antibody's specificity would better suit your research question.

• Conjugation: Determine if you need an unconjugated antibody or one conjugated to a reporter molecule (HRP, FITC, biotin) depending on your detection system .

Review product datasheets and published literature to confirm the antibody's performance in experimental conditions similar to yours.

How can SLC25A37 antibodies be used to study iron metabolism in cancer progression?

SLC25A37 antibodies provide valuable tools for investigating the role of iron metabolism in cancer progression, particularly in the context of metastasis. Recent CRISPR screening has identified SLC25A37 as an organ-specific requirement for liver metastasis in breast cancer models . To study this phenomenon:

• Expression analysis: Use SLC25A37 antibodies for Western blot and IHC to compare expression levels between primary tumors and metastatic lesions from different organs. This approach revealed that SLC25A37 is highly expressed in liver compared to lung metastases of breast cancer patients .

• Mechanistic studies: Combine SLC25A37 antibody detection with hypoxia markers to investigate the relationship between HIF1α signaling and SLC25A37 expression, as research has shown that SLC25A37 expression is induced by HIF1α to support heme synthesis in hypoxic liver regions .

• Therapeutic response assessment: Use flow cytometry with SLC25A37 antibodies to evaluate how iron chelation or ferroptosis-inducing therapies affect cancer cell populations with varying SLC25A37 expression levels. This is particularly relevant since SLC25A37-dependent bilirubin synthesis has been identified as a requirement for liver metastasis to resist ferroptosis .

• Co-localization studies: Employ dual immunofluorescence with SLC25A37 antibodies and mitochondrial markers to investigate changes in mitochondrial iron transport during cancer progression and metastasis.

These approaches can provide insights into how iron metabolism adaptations contribute to cancer cells' ability to colonize specific organ microenvironments.

What are the considerations when using SLC25A37 antibodies for studying tissue-specific iron homeostasis?

When investigating tissue-specific iron homeostasis using SLC25A37 antibodies, researchers should consider several important factors:

• Tissue expression heterogeneity: SLC25A37 expression varies significantly between tissues, with particularly important roles in erythroid cells and liver tissue. When designing immunohistochemistry experiments, include appropriate positive control tissues and optimize staining protocols for each tissue type .

• Subcellular localization confirmation: As a mitochondrial inner membrane protein, SLC25A37 should co-localize with mitochondrial markers. Use co-immunofluorescence with established mitochondrial markers to confirm proper localization and antibody specificity .

• Physiological state considerations: Iron homeostasis is dynamic and responsive to systemic conditions. Document and control for variables that might affect SLC25A37 expression, such as hypoxia, inflammation, or iron availability in the experimental system .

• Isoform specificity: SLC25A37 has multiple isoforms produced by alternative splicing . Determine which isoform(s) your antibody recognizes and whether these isoforms have tissue-specific expression patterns. Some research has shown higher expression of specific SLC25A37 isoforms in certain disease states, such as in SF3B1-mutant patients .

• Comparative analysis approach: When comparing SLC25A37 expression across tissues, use standardized protein quantification methods and loading controls specific for mitochondrial proteins to accurately assess relative expression levels.

Careful consideration of these factors will enhance the validity and interpretability of results when studying tissue-specific aspects of iron homeostasis using SLC25A37 antibodies.

What are the optimal conditions for Western blot analysis using SLC25A37 antibodies?

Achieving optimal Western blot results with SLC25A37 antibodies requires careful attention to several methodological factors:

• Sample preparation:

  • For cellular samples: Use RIPA buffer supplemented with protease inhibitors to extract total protein, including membrane-bound mitochondrial proteins .

  • For tissue samples: Homogenization in tissue-specific lysis buffers is critical; for example, spleen tissue (rich in iron-handling cells) requires special attention to prevent protein degradation during extraction .

  • Include mitochondrial isolation step when focusing specifically on mitochondrial proteins to enrich for SLC25A37.

• Protein loading and separation:

  • Load 25-35 μg of total protein per lane (as used in validated protocols) .

  • Use 10-12% SDS-PAGE gels for optimal resolution of SLC25A37, which has a calculated molecular weight of approximately 37 kDa .

• Transfer conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for hydrophobic mitochondrial membrane proteins).

  • Use wet transfer systems at lower voltage (30V) for longer duration (2 hours) to ensure efficient transfer of membrane proteins.

• Blocking and antibody conditions:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

  • Dilute primary SLC25A37 antibody at 1:1000 to 1:2000 in blocking buffer and incubate overnight at 4°C .

  • Wash thoroughly (4-5 times) with TBST before and after secondary antibody incubation.

  • Use anti-rabbit HRP-conjugated secondary antibody at 1:5000 to 1:10000 dilution for 1 hour at room temperature.

• Controls:

  • Include positive control samples with known SLC25A37 expression (K562 cell line, rat spleen tissue) .

  • Consider using siRNA or CRISPR knockout samples as negative controls to validate antibody specificity.

These optimized conditions have been validated in published protocols and should provide specific detection of SLC25A37 protein in Western blot applications.

How should I optimize immunohistochemistry protocols for SLC25A37 detection in different tissue types?

Optimizing immunohistochemistry (IHC) protocols for SLC25A37 detection across different tissue types requires systematic adjustment of several parameters:

• Tissue fixation and processing:

  • For formalin-fixed paraffin-embedded (FFPE) tissues: Standard 10% neutral buffered formalin fixation for 24-48 hours is suitable, but overfixation can mask epitopes .

  • For mitochondrial proteins like SLC25A37, consider using Zenker's or Bouin's fixatives which better preserve mitochondrial structures in tissues with high metabolic activity.

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is generally effective for SLC25A37 antibodies .

  • For tissues with high iron content (liver, spleen), add a mild chelating agent to the retrieval buffer to prevent interference from endogenous iron.

  • Optimize retrieval time: 15-20 minutes for most tissues, potentially longer for dense tissues like liver.

• Tissue-specific considerations:

  • Brain tissue: Requires longer permeabilization steps due to lipid content; use 0.3% Triton X-100 in PBS for 15 minutes .

  • Liver tissue: Contains high endogenous peroxidase activity; use 3% hydrogen peroxide for 15 minutes followed by additional blocking with 0.1% sodium azide.

  • Hematopoietic tissues: May require additional blocking steps with serum matched to the secondary antibody species.

• Antibody dilution and incubation:

  • Start with manufacturer's recommended dilution and optimize through titration experiments for each tissue type.

  • For tissues with expected low expression, use higher antibody concentration and longer incubation times (overnight at 4°C).

  • For tissues with high SLC25A37 expression, dilute antibody further to prevent background.

• Detection systems:

  • For tissues with high endogenous biotin (liver, kidney): Avoid biotin-based detection systems and opt for polymer-based detection.

  • For tissues with high autofluorescence: Use brightfield IHC with DAB substrate rather than immunofluorescence.

• Validation controls:

  • Include tissue-matched positive and negative controls in each experimental run.

  • Consider dual staining with mitochondrial markers to confirm subcellular localization.

By systematically optimizing these parameters for each tissue type, researchers can achieve consistent and specific SLC25A37 detection across diverse tissue samples.

Why might I be observing inconsistent SLC25A37 antibody staining in my immunofluorescence experiments?

Inconsistent staining in immunofluorescence experiments with SLC25A37 antibodies can stem from several experimental factors:

• Cell fixation issues:

  • SLC25A37 is a mitochondrial membrane protein, and improper fixation can disrupt mitochondrial morphology and epitope accessibility.

  • Solution: Optimize fixation by testing 4% paraformaldehyde (10-15 minutes) versus methanol fixation (-20°C, 10 minutes). Methanol often better preserves mitochondrial structures .

  • Avoid overfixation, which can mask epitopes and create false negatives.

• Permeabilization problems:

  • Insufficient permeabilization prevents antibody access to mitochondrial inner membrane targets.

  • Solution: Test different permeabilization conditions (0.1-0.5% Triton X-100 for 5-10 minutes or 0.1% saponin) to optimize for mitochondrial membrane access without destroying mitochondrial integrity .

• Epitope masking due to protein interactions:

  • SLC25A37 function depends on protein-protein interactions which may mask epitopes in certain cellular states.

  • Solution: Try antibodies targeting different epitopes (N-terminal vs C-terminal) as accessibility may vary in different experimental conditions .

• Heterogeneous expression:

  • SLC25A37 expression varies with cellular iron requirements and metabolic state.

  • Solution: Synchronize cells or control for experimental conditions that affect iron metabolism; consider co-staining with iron-regulatory proteins to correlate with functional states.

• Technical considerations:

  • Proper antibody dilution is critical - too concentrated leads to background, too dilute yields weak signals.

  • Solution: Perform antibody titration (1:25 to 1:500) to determine optimal concentration for your specific cell type .

  • Include appropriate controls: positive control cells with known SLC25A37 expression (e.g., K562 cells) and negative controls (secondary antibody only).

• Mitochondrial dynamics:

  • Mitochondria constantly undergo fusion and fission, affecting the distribution of membrane proteins.

  • Solution: Consider fixation timing relative to cell cycle or treatments that affect mitochondrial dynamics.

Implementing these troubleshooting approaches systematically can help achieve more consistent immunofluorescence staining patterns for SLC25A37.

How can I validate SLC25A37 antibody specificity for my research?

Validating antibody specificity is crucial for ensuring reliable research results. For SLC25A37 antibodies, implement these comprehensive validation strategies:

• Genetic knockdown/knockout validation:

  • Perform siRNA knockdown or CRISPR/Cas9 knockout of SLC25A37 in your experimental system.

  • Compare antibody staining between wild-type and knockdown/knockout samples across all applications (Western blot, IHC, IF) to confirm signal reduction or elimination in genetic models .

• Overexpression systems:

  • Create transient or stable overexpression of tagged SLC25A37 constructs.

  • Confirm co-localization of antibody signal with the tagged protein.

  • This approach is particularly useful for validating antibodies against low-abundance targets.

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (if available from manufacturer).

  • A specific antibody will show reduced or absent signal when pre-blocked with its target peptide .

• Multi-antibody validation:

  • Test multiple antibodies targeting different epitopes of SLC25A37 (N-terminal, C-terminal, and middle regions) .

  • Consistent detection patterns across different antibodies increase confidence in specificity.

• Cross-species reactivity assessment:

  • If your antibody claims cross-reactivity with multiple species, test samples from each species.

  • Compare detection patterns with expected evolutionary conservation of the protein.

• Subcellular localization confirmation:

  • As a mitochondrial inner membrane protein, SLC25A37 should co-localize with established mitochondrial markers.

  • Perform dual immunofluorescence with antibodies against mitochondrial markers (e.g., TOMM20, COX IV) to confirm proper localization .

• Mass spectrometry validation:

  • For advanced validation, perform immunoprecipitation with the SLC25A37 antibody followed by mass spectrometry analysis.

  • This identifies all proteins captured by the antibody and confirms whether SLC25A37 is the predominant target.

Documenting these validation steps provides strong evidence for antibody specificity, enhancing the reliability and reproducibility of your research findings.

How can SLC25A37 antibodies be utilized to study the role of iron metabolism in cancer metastasis?

Recent research has identified SLC25A37 as a critical factor in organ-specific cancer metastasis, particularly in liver metastasis. Researchers can use SLC25A37 antibodies to investigate this phenomenon through several methodological approaches:

• Comparative expression analysis:

  • Use immunohistochemistry with validated SLC25A37 antibodies to compare expression between primary tumors and their metastases across different organs.

  • Create tissue microarrays of patient samples to quantitatively assess expression differences between lung and liver metastases, as research has shown SLC25A37 is highly expressed in liver compared to lung metastases of breast cancer patients .

• Mechanistic pathway investigations:

  • Employ co-immunoprecipitation with SLC25A37 antibodies to identify interaction partners in metastatic versus non-metastatic cancer cells.

  • Combine with Western blot analysis of iron regulatory proteins to establish connections between iron metabolism and metastatic potential.

• Functional studies:

  • Use flow cytometry with SLC25A37 antibodies to sort cancer cells based on expression levels.

  • Correlate expression with functional assays measuring iron uptake, heme synthesis, and resistance to ferroptosis.

  • Track sorted populations for their metastatic potential in animal models.

• Therapeutic response assessment:

  • Apply SLC25A37 antibodies in immunofluorescence to monitor protein expression changes in response to iron chelation therapy or ferroptosis inducers.

  • Develop tissue culture models where cancer cells are exposed to liver-specific microenvironmental factors to assess how these influence SLC25A37 expression and function.

• Hypoxia response evaluation:

  • Use dual staining with SLC25A37 and HIF1α antibodies to investigate the relationship between hypoxia and SLC25A37 expression in tumor samples.

  • This approach can validate findings that SLC25A37 expression is induced by HIF1α to support heme synthesis, enabling cancer cells to grow in hypoxic liver regions through production of the lipophilic antioxidant bilirubin .

These methodological approaches provide a framework for investigating how iron metabolism adaptations, particularly through SLC25A37, contribute to the organ-specific nature of cancer metastasis.

What experimental considerations are important when using SLC25A37 antibodies in ferroptosis research?

Ferroptosis, an iron-dependent form of regulated cell death, is increasingly recognized as relevant to cancer biology. When using SLC25A37 antibodies in ferroptosis research, consider these critical experimental parameters:

• Cell model selection and validation:

  • Choose cell models with varied SLC25A37 expression levels to establish correlations with ferroptosis sensitivity.

  • Validate SLC25A37 expression in your models using Western blot before proceeding with functional experiments .

  • Consider cells derived from liver metastases, as research indicates SLC25A37-dependent pathways help these cells resist ferroptosis .

• Experimental timing considerations:

  • Monitor SLC25A37 expression dynamics during ferroptosis induction using time-course immunoblotting or immunofluorescence.

  • Consider that mitochondrial morphology changes during ferroptosis, potentially affecting antibody accessibility to SLC25A37 epitopes.

• Functional correlations:

  • Combine SLC25A37 antibody detection with assays measuring:

    • Mitochondrial iron content (using mitochondrial isolation followed by iron quantification)

    • Lipid peroxidation (BODIPY-C11 staining)

    • Glutathione levels (indicator of antioxidant capacity)

    • Bilirubin production (identified as a downstream effector of SLC25A37 in ferroptosis resistance)

• Induction and inhibition protocols:

  • When treating cells with ferroptosis inducers (e.g., erastin, RSL3), monitor SLC25A37 expression changes.

  • In parallel experiments, use ferroptosis inhibitors (e.g., ferrostatin-1, liproxstatin-1) to determine if SLC25A37 expression changes are specific to the ferroptotic process.

  • Consider that research has shown treating mice with ferroptosis inhibitors fully restored the capacity of SLC25A37-deficient cancer cells to grow in the liver .

• Subcellular localization assessment:

  • During ferroptosis, mitochondrial morphology and membrane integrity change dramatically.

  • Use co-immunofluorescence with markers for mitochondrial outer membrane, intermembrane space, and matrix to track potential relocalization of SLC25A37 during ferroptosis progression.

• Ex vivo and in vivo translation:

  • Validate findings from cell culture in tissue samples from animal models treated with ferroptosis inducers/inhibitors.

  • Use immunohistochemistry with SLC25A37 antibodies on tissue sections from these models to correlate expression with treatment response.

These methodological considerations will enhance the rigor and reproducibility of research investigating the role of SLC25A37 in ferroptosis resistance, particularly in the context of cancer metastasis.

What are the most effective protein extraction methods for detection of SLC25A37 in Western blot analysis?

Efficient extraction of SLC25A37, a mitochondrial inner membrane protein, requires specialized protocols to ensure complete solubilization while maintaining protein integrity:

• Total protein extraction protocol:

  • Buffer composition: Use RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with:

    • Protease inhibitor cocktail (AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A)

    • Phosphatase inhibitors (sodium fluoride, sodium orthovanadate)

    • 1 mM PMSF (add fresh)

  • Extraction procedure:

    • For cells: Add 500 μl cold RIPA buffer per 10^7 cells, incubate on ice for 30 minutes with vortexing every 10 minutes

    • For tissues: Homogenize 50-100 mg tissue in 1 ml cold RIPA buffer using a tissue homogenizer

    • Centrifuge at 14,000 × g for 15 minutes at 4°C

    • Collect supernatant containing solubilized proteins

• Enriched mitochondrial fraction protocol:

  • For enhanced detection sensitivity, isolate mitochondria before protein extraction:

    • Homogenize cells/tissues in mitochondrial isolation buffer (225 mM mannitol, 75 mM sucrose, 10 mM HEPES, 1 mM EGTA, pH 7.4)

    • Perform differential centrifugation: 600 × g (10 min) to remove nuclei and debris, then 7,000 × g (10 min) to pellet mitochondria

    • Resuspend mitochondrial pellet in RIPA buffer for protein extraction

  • This approach concentrates mitochondrial proteins, improving detection of less abundant proteins like SLC25A37

• Sample handling considerations:

  • Maintain samples at 4°C throughout extraction to prevent proteolysis

  • Avoid repeated freeze-thaw cycles which can degrade membrane proteins

  • When preparing samples for gel loading, heat at 70°C (not 95°C) for 10 minutes to prevent aggregation of membrane proteins

  • Add reducing agent (β-mercaptoethanol or DTT) to disrupt potential disulfide bonds

• Sample quantification:

  • Use BCA or Bradford assay for protein quantification

  • Load 25-35 μg of total protein or 10-15 μg of mitochondrial fraction for optimal detection

  • Include positive control samples with known SLC25A37 expression (K562 cell line or rat spleen tissue lysates have been validated)

These optimized extraction methods ensure reliable and consistent detection of SLC25A37 in Western blot applications across different experimental systems.

How can I optimize dual immunofluorescence staining to co-localize SLC25A37 with other mitochondrial markers?

Optimizing dual immunofluorescence to co-localize SLC25A37 with other mitochondrial markers requires careful attention to experimental design and protocol optimization:

• Antibody selection and validation:

  • Primary antibody compatibility: Select SLC25A37 antibody and mitochondrial marker antibodies (e.g., TOMM20, COX IV, MitoTracker) raised in different host species to avoid cross-reactivity .

  • Validate each antibody individually before attempting co-staining to establish optimal dilutions and confirm specific staining patterns.

  • Consider using directly conjugated antibodies when available to simplify protocols and reduce background.

• Sample preparation optimization:

  • Fixation method: Test both paraformaldehyde (4%, 10-15 minutes) and methanol (-20°C, 10 minutes) fixation to determine which better preserves both antigens.

  • Permeabilization: Optimize detergent concentration and incubation time (typically 0.1-0.3% Triton X-100 for 5-10 minutes) to ensure antibody access to mitochondrial membranes without destroying mitochondrial morphology.

  • Blocking: Use 5-10% normal serum from the species of secondary antibodies combined with 1% BSA to minimize nonspecific binding.

• Sequential staining protocol:

  • Primary antibody incubation:

    • Option 1: Apply antibodies sequentially (incubate with first primary antibody, wash, then apply second primary)

    • Option 2: Apply antibodies simultaneously if raised in different species and validated for compatibility

    • Dilute SLC25A37 antibody at 1:25 to 1:100 as reported in validated protocols

  • Secondary antibody selection:

    • Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity

    • Select fluorophores with well-separated emission spectra (e.g., Alexa Fluor 488 and Alexa Fluor 594)

    • Include appropriate controls (secondary-only, single primary) to assess bleed-through and cross-reactivity

• Image acquisition considerations:

  • Acquire images sequentially (not simultaneously) using confocal microscopy to minimize bleed-through

  • Optimize exposure settings independently for each channel

  • Use the same acquisition parameters across all experimental conditions for valid comparisons

• Quantitative co-localization analysis:

  • Employ Pearson's or Mander's coefficient calculations to quantify the degree of co-localization

  • Use specialized image analysis software (ImageJ with Coloc2 plugin, CellProfiler) for unbiased quantification

  • Analyze at least 50-100 cells per condition for statistical validity

• Troubleshooting strategies:

  • If signals interfere: Try using Fab fragments instead of full IgG secondary antibodies

  • If signal is weak for one antibody: Perform tyramide signal amplification for the weaker signal

  • If mitochondrial morphology is poor: Test milder permeabilization methods (digitonin instead of Triton X-100)