slc25a37 Antibody

What is SLC25A37 and why is it important in scientific research?

SLC25A37 (Mitoferrin-1 or MFRN1) is a mitochondrial iron transporter that belongs to the SLC25 family of mitochondrial carrier proteins. The canonical human protein has 338 amino acid residues with a molecular weight of approximately 37.3 kDa and is localized to the mitochondria . SLC25A37 plays a critical role in shuttling ferrous iron into the mitochondrial matrix, where it is primarily used for heme synthesis and iron-sulfur cluster formation .

Recent research has demonstrated that SLC25A37 is particularly important in hypoxic environments and is directly regulated by HIF1α, making it crucial for understanding cellular adaptations to low oxygen conditions, especially in cancer metastasis research . The protein has been identified as an organ-specific dependency in liver metastasis but not lung metastasis, highlighting its potential significance as a therapeutic target in specific cancer contexts .

What applications are SLC25A37 antibodies typically validated for?

SLC25A37 antibodies have been validated for multiple research applications, with different antibodies showing varying performance across techniques:

When designing experiments, researchers should consider the specific epitope targeted by the antibody, as some antibodies are raised against the C-terminal region while others may target different regions of the protein.

How should I select the appropriate SLC25A37 antibody for my specific research question?

Selecting the appropriate SLC25A37 antibody requires consideration of several factors:

• Target Species: Verify cross-reactivity with your experimental model. Some antibodies react only with human SLC25A37, while others cross-react with mouse, rat, or other species .

• Application Compatibility: Review validation data for your intended application. For example, if performing co-localization studies in mitochondria, select antibodies validated for immunofluorescence with demonstrated mitochondrial localization .

• Epitope Location: Consider whether the epitope is accessible in your experimental conditions. For membrane proteins like SLC25A37, antibodies targeting extracellular domains may be preferable for non-permeabilized applications .

• Antibody Format: For multi-color imaging or flow cytometry, consider conjugated antibodies or ensure your antibody is compatible with secondary detection systems .

• Clone Type: Monoclonal antibodies offer higher specificity and reproducibility while polyclonal antibodies might provide higher sensitivity but more batch-to-batch variation .

What are the optimal fixation and permeabilization conditions for detecting SLC25A37 in different sample types?

Optimal detection of SLC25A37 in different sample types requires appropriate fixation and permeabilization protocols:

For Immunofluorescence and Immunocytochemistry:

• Paraformaldehyde (4%) fixation for 10-15 minutes at room temperature works well for most cell lines, as demonstrated in HeLa cells with SLC25A37 antibodies .

• Since SLC25A37 is a mitochondrial protein, thorough permeabilization is essential. A combination of 0.1-0.2% Triton X-100 for 10 minutes is typically effective.

• Some protocols benefit from methanol permeabilization (-20°C for 10 minutes) which can better expose mitochondrial epitopes.

For Immunohistochemistry:

• Formalin-fixed paraffin-embedded (FFPE) tissues have been successfully used with SLC25A37 antibodies at dilutions of 1:10-1:50 or 1:200-1:500 depending on the antibody .

• Antigen retrieval is critical - citrate buffer (pH 6.0) heat-induced epitope retrieval has proven effective for many mitochondrial proteins including SLC25A37.

• Brain tissue sections have been successfully stained using DAB visualization methods following peroxidase conjugation of secondary antibodies .

For hypoxic tissue samples (particularly relevant for SLC25A37 research), special attention should be paid to rapid fixation to preserve the hypoxic state and protein modification status.

How can I validate the specificity of SLC25A37 antibodies in my experimental system?

Validating antibody specificity is crucial for reliable SLC25A37 research:

• Genetic Controls: The gold standard for validation is using CRISPR/Cas9 knockout or shRNA knockdown samples. Research has successfully employed lentiviral vectors containing sgRNAs against SLC25A37 or non-targeting controls to validate antibody specificity .

• Peptide Competition: Pre-incubating the antibody with the immunizing peptide should abolish specific staining. Some suppliers offer blocking peptides specifically for SLC25A37 antibody validation .

• Multiple Antibodies: Using antibodies raised against different epitopes of SLC25A37 should yield consistent localization and molecular weight patterns.

• Expected Localization: SLC25A37 should co-localize with mitochondrial markers. Validation can include co-staining with established mitochondrial markers like MitoTracker or TOM20.

• Molecular Weight Verification: Western blot should detect a band at approximately 37.3 kDa, the predicted molecular weight for SLC25A37 .

• Cross-Species Reactivity: If the antibody claims cross-reactivity, verify consistent patterns across species using similar tissue types.

One study validated anti-SLC25A37 antibodies by comparing expression patterns in control K562 cell lines and rat spleen tissue lysates, confirming appropriate molecular weight detection .

What are common pitfalls in SLC25A37 antibody-based detection and how can they be overcome?

Several challenges can affect SLC25A37 antibody-based detection:

• Mitochondrial Specificity: The mitochondrial localization of SLC25A37 can make it difficult to distinguish from other mitochondrial proteins.

  • Solution: Super-resolution microscopy or proximity ligation assays can help resolve co-localization with other mitochondrial markers.

• Cross-Reactivity with SLC25A28 (MFRN2): SLC25A37 (MFRN1) has a paralog, SLC25A28 (MFRN2), with similar sequence and function.

  • Solution: Verify antibody specificity using cells with genetic manipulation of each paralog separately. One study demonstrated this using SpCas9-competent cell lines expressing sgRNAs targeting either MFRN1 or MFRN2 .

• Hypoxia-Induced Expression Changes: SLC25A37 expression is regulated by hypoxia through HIF1α, potentially affecting detection levels .

  • Solution: Standardize oxygen conditions during sample preparation and consider using HIF1α staining as an internal control for hypoxic regions.

• Tissue-Specific Expression Levels: Expression varies significantly between tissues (higher in liver versus lung metastases) .

  • Solution: Adjust antibody concentrations based on expected expression levels and include positive control tissues.

• Fixation-Sensitive Epitopes: Some epitopes may be sensitive to over-fixation.

  • Solution: Optimize fixation time and consider comparing multiple fixation methods (PFA, methanol, acetone) for your specific antibody.

How can SLC25A37 antibodies be optimized for detecting differences in hypoxic versus normoxic tissues?

Given that SLC25A37 is regulated by hypoxia through HIF1α binding to its promoter , optimizing detection in different oxygen conditions requires specific approaches:

• Antibody Selection: Select antibodies that target epitopes unlikely to be affected by hypoxia-induced post-translational modifications. C-terminal antibodies have shown consistent results across oxygen conditions .

• Normalization Strategy:

  • Include HIF1α staining as a positive control for hypoxic regions

  • Use quantitative approaches like mean fluorescence intensity ratios of SLC25A37 to mitochondrial markers

• Experimental Controls:

  • Compare tissues from well-perfused versus poorly-perfused regions (which can be identified using Hoechst dye injection prior to tissue collection)

  • Include parallel samples from normoxic cultures as baseline references

• Multiplexed Detection: Combine SLC25A37 antibodies with antibodies against canonical hypoxia response genes like CA9, BNIP3, and GLUT1, which have been used to validate hypoxic conditions in tissue sections .

• Signal Amplification: For tissues with low expression under normoxic conditions, consider using signal amplification methods such as tyramide signal amplification to detect subtle differences in expression levels.

Research has shown that SLC25A37 induction is particularly strong in liver compared to lung metastases under hypoxic conditions, with significant HIF1α binding to the SLC25A37 promoter detected by ChIP-qPCR .

What approaches are recommended for multiplexed detection of SLC25A37 with other iron metabolism proteins?

For comprehensive analysis of iron metabolism pathways, multiplexed detection of SLC25A37 with other related proteins provides valuable insights:

• Compatible Antibody Selection:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-SLC25A37 with mouse anti-ferrochelatase)

  • Alternatively, use directly conjugated antibodies with distinct fluorophores

• Recommended Protein Combinations:

  • SLC25A37 + Ferrochelatase (FECH) - Studies have shown functional relationships between these proteins in heme biosynthesis

  • SLC25A37 + ABCB10 - These proteins form an oligomeric complex that stabilizes SLC25A37

  • SLC25A37 + Heme Oxygenase (HO-1) - HO-1 showed reduced stability upon SLC25A37 loss

  • SLC25A37 + Biliverdin Reductase (BLVRA) - For studying the complete heme catabolism pathway

• Multiplex Optimization:

  • Sequential staining protocols may be necessary to avoid cross-reactivity

  • Carefully titrate each antibody to minimize background

  • Include appropriate single-stained controls for spectral unmixing

• Advanced Readout Methods:

  • Consider mass cytometry (CyTOF) for highly multiplexed protein detection

  • Some SLC25A37 antibodies have been validated in cytometric bead arrays for multiplexed detection

• Proximity-Based Assays:

  • Proximity ligation assays (PLA) can detect protein-protein interactions between SLC25A37 and other iron metabolism proteins with subcellular resolution

Research has demonstrated the close functional relationship between SLC25A37, heme catabolism, and bilirubin synthesis in vivo, making these multiplexed approaches particularly valuable .

How should SLC25A37 antibody staining patterns be interpreted in the context of cancer research?

Interpreting SLC25A37 antibody staining in cancer tissues requires consideration of several contexts:

• Subcellular Localization:

  • Normal pattern: Punctate mitochondrial staining

  • Abnormal patterns: Diffuse cytoplasmic staining may indicate mitochondrial dysfunction

• Tissue-Specific Expression:

  • SLC25A37 shows higher expression in liver compared to lung metastases

  • Expression correlates with hypoxic regions, particularly in poorly perfused areas of liver metastases

• Correlation with Hypoxia Markers:

  • Co-expression with HIF1α suggests hypoxia-driven upregulation

  • Research has demonstrated stronger induction of SLC25A37 and HIF1α protein in liver compared to lung metastases

• Heterogeneity Analysis:

  • Quantify expression across different tumor regions using image analysis tools

  • Compare expression between invasive fronts versus tumor cores

  • Assess correlation with distance from blood vessels

• Prognostic/Predictive Interpretation:

  • High SLC25A37 expression in liver metastases may indicate adaptation to hypoxia

  • Loss of SLC25A37 has been shown to reduce liver metastasis formation in mouse models

A scoring system combining intensity (0-3) and percentage of positive cells can be used for semi-quantitative assessment. Research has shown that SLC25A37 expression is particularly relevant in hypoxic regions of liver metastases, where it supports heme synthesis enabling cancer cells to produce the lipophilic antioxidant bilirubin that protects against ferroptosis .

What controls are necessary for accurate quantification of SLC25A37 expression using immunohistochemistry or immunofluorescence?

Accurate quantification of SLC25A37 expression requires comprehensive controls:

For advanced quantification, consider:

• Z-stack acquisitions for 3D volume analysis of mitochondrial expression

• Single-cell analysis approaches to capture heterogeneity

• Multi-parameter scoring incorporating intensity, percentage positive cells, and subcellular localization

Research has employed flow cytometry-based isolation of cancer cells from different tissue regions (using CD90.1 tagging) followed by RT-qPCR to accurately quantify SLC25A37 expression differences between high and low perfused regions , providing a complementary approach to direct antibody-based detection.