sfxn4 Antibody

What is SFXN4 and why is it important in cellular research?

SFXN4 (Sideroflexin 4) is a highly conserved mitochondrial inner membrane protein that belongs to the sideroflexin family. Initially characterized as a potential iron transporter, recent research has revealed its critical role in iron-sulfur (Fe-S) cluster biogenesis and mitochondrial respiratory chain complex I assembly .

SFXN4 has gained significant research attention because:

• It functions as a complex I assembly factor by interacting with the MCIA (Mitochondrial Complex I Assembly) complex and is required for the assembly of the ND2 module

• It plays a role in iron homeostasis and mitochondrial function

• Its inhibition causes accumulation of excess iron leading to oxidative stress in cancer cells

• It impacts DNA repair mechanisms by affecting Fe-S-dependent DNA repair enzymes

• Mutations in SFXN4 are associated with mitochondrial disease

This protein represents a fascinating evolutionary story, as other sideroflexin family members (SFXN1/2/3) function as mitochondrial serine transporters, indicating SFXN4 has evolved a distinct function .

What are the optimal dilutions and applications for SFXN4 antibodies?

Based on multiple commercial antibody datasheets and research applications, the following optimal working dilutions are recommended:

The optimal dilution should be determined empirically by each researcher, as factors such as sample type, protein abundance, and specific antibody characteristics can influence results .

For immunofluorescence applications, SFXN4 antibodies have been valuable for confirming mitochondrial localization with a characteristic punctate staining pattern that co-localizes with mitochondrial markers .

How can researchers validate the specificity of SFXN4 antibodies?

Validating antibody specificity is crucial for generating reliable research data. For SFXN4 antibodies, researchers should consider these validation approaches:

• Genetic validation: Use SFXN4 knockout (KO) cell lines as negative controls. Multiple studies have utilized CRISPR-Cas9 generated SFXN4 KO cells to confirm antibody specificity .

• Rescue experiments: Re-express SFXN4 in knockout cells (e.g., using SFXN4KO + FLAGSFXN4 constructs) to restore detection and confirm specificity .

• siRNA knockdown: Perform siRNA-mediated knockdown (as demonstrated in HCC studies) to show reduced antibody signal intensity correlating with reduced protein levels .

• Molecular weight verification: Confirm detection at the expected molecular weight (37-38kDa for human SFXN4) .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding.

• Cross-reactivity assessment: Test reactivity against related proteins (other SFXN family members) to ensure specificity within the sideroflexin family.

Researchers should document these validation steps in their publications to increase research reproducibility .

What are the best approaches for studying SFXN4 interactions with the MCIA complex?

Studying the interaction between SFXN4 and the MCIA complex requires specialized techniques:

• Co-immunoprecipitation (Co-IP): Use anti-FLAG immunoprecipitation with tagged SFXN4 constructs (FLAGSFXN4) or reciprocal IP with tagged MCIA components (e.g., ACAD9FLAG). This approach has successfully demonstrated physical interactions between SFXN4 and MCIA components like ECSIT, ACAD9, and NDUFAF1 .

• Blue Native PAGE (BN-PAGE): This technique is essential for preserving native protein complexes. BN-PAGE followed by immunoblotting has revealed SFXN4-containing complexes that comigrate with MCIA components. Key findings include:

  • A SFXN4-MCIA complex migrating at a specific molecular weight that is absent in SFXN4 KO cells

  • Higher molecular weight complexes representing more advanced assembly intermediates containing both ND2 and ND1 modules

• Complexome profiling: This MS-based proteomics technique profiles the composition and size of protein complexes separated by BN-PAGE. It has revealed extensive rearrangements of MCIA complex intermediates in SFXN4 KO cells .

• Affinity enrichment mass spectrometry (AP-MS): This approach identified the three most enriched SFXN4-interacting proteins as ECSIT, ACAD9, and NDUFAF1 - established assembly factors for complex I .

• Gene essentiality correlation analysis: Using the Achilles gene essentiality dataset can provide strong evidence for functionally relevant protein interactions .

These techniques should be used in combination for comprehensive characterization of SFXN4-MCIA interactions.

How can SFXN4 antibodies be utilized to investigate iron-sulfur cluster biogenesis in cancer research?

SFXN4 antibodies are valuable tools for investigating the role of iron-sulfur (Fe-S) cluster biogenesis in cancer, particularly through these methodological approaches:

• Expression correlation studies: Use SFXN4 antibodies for immunohistochemistry on cancer tissue microarrays to correlate expression levels with clinical outcomes. Research has shown SFXN4 is upregulated in hepatocellular carcinoma (HCC) and correlates with poor prognosis .

• Functional studies in knockdown models:

  • Western blot with SFXN4 antibodies to confirm successful protein depletion

  • Measure Fe-S-dependent enzyme activities after SFXN4 knockdown

  • Assess iron accumulation and oxidative stress markers

• Therapeutic sensitivity assessment: SFXN4 knockdown sensitizes ovarian cancer cells to DNA-damaging drugs and PARP inhibitors through dual mechanisms:

  • Inhibition of Fe-S biogenesis triggers iron accumulation and oxidative stress

  • Impairment of Fe-S-dependent DNA repair enzymes

• Protein-protein interaction studies: Use co-immunoprecipitation with SFXN4 antibodies to identify cancer-specific interaction partners involved in Fe-S biogenesis .

• Subcellular localization: Immunofluorescence with SFXN4 antibodies can reveal altered mitochondrial morphology and distribution in cancer cells, particularly after treatments affecting iron metabolism .

These approaches have revealed that SFXN4 may represent a promising new target in ovarian cancer therapy, as its inhibition sensitizes even drug-resistant ovarian cancer cells to common treatments .

What sample preparation methods are recommended for SFXN4 antibody-based experiments?

Appropriate sample preparation is crucial for SFXN4 detection due to its mitochondrial localization and involvement in protein complexes:

• For Western blotting:

  • Mitochondrial isolation: For enriched detection, isolate mitochondria using differential centrifugation or commercial isolation kits

  • Protein extraction: Use buffers containing 1% digitonin for preserving protein-protein interactions or RIPA buffer for total protein extraction

  • Sample heating: Heat samples at 70°C rather than boiling to prevent aggregation of membrane proteins

  • Reducing conditions: Include DTT or β-mercaptoethanol in sample buffer

• For immunoprecipitation studies:

  • Solubilize mitochondria in 1% digitonin-containing buffer to maintain native complexes

  • Use anti-FLAG IP for tagged constructs (SFXN4KO + FLAGSFXN4) to study interaction partners

• For native complex analysis (BN-PAGE):

  • Solubilize mitochondria in 1% digitonin

  • Avoid harsh detergents that disrupt protein-protein interactions

  • Include Coomassie blue G-250 in the sample buffer

• For immunofluorescence:

  • Fixation: 4% paraformaldehyde (10-15 minutes)

  • Permeabilization: 0.1-0.2% Triton X-100

  • Co-staining with mitochondrial markers (e.g., MitoTracker, TOM20) to confirm localization

  • Optimal antibody dilution range: 1:50-1:200

• For immunohistochemistry:

  • Both paraffin-embedded and frozen sections can be used

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Recommended dilution: 1:20-1:200

Proper controls, including SFXN4 knockout or knockdown samples, should be included in all experiments to confirm specificity .

What considerations should be made when using SFXN4 antibodies for cancer biomarker research?

When investigating SFXN4 as a potential cancer biomarker, researchers should consider:

• Expression analysis across cancer types:

  • SFXN4 is consistently elevated in hepatocellular carcinoma (HCC) and correlates with poor outcomes

  • Comprehensive analysis across multiple cancer datasets is recommended to establish tissue-specific expression patterns

• Correlation with clinicopathological features:

  • SFXN4 expression positively correlates with clinicopathological characteristics in HCC

  • Document tumor stage, grade, and patient survival data alongside SFXN4 expression

• Multivariate analysis with other biomarkers:

  • Combine SFXN4 with established cancer markers for improved prognostic value

  • Consider analyzing all sideroflexin family members (SFXN1-5) for comprehensive profiling

• Functional validation in cellular models:

  • Knockdown studies show SFXN4 inhibition decreases proliferation, migration and invasion in HCC cells

  • Expression of cell cycle markers (cyclin D1) and matrix metalloproteinases (MMP2) are reduced after SFXN4 knockdown

  • In vivo tumor xenograft models confirm growth inhibition with SFXN4 knockdown

• Mechanism investigation:

  • Bioinformatic analysis has revealed SFXN4 is primarily involved in:

    • Oxidative phosphorylation

    • Reactive oxygen species metabolism

    • Metabolic pathways

  • SFXN4 expression correlates with immune infiltration in HCC, suggesting immune-related functions

• Therapeutic relevance:

  • In ovarian cancer, SFXN4 inhibition sensitizes cells to cisplatin and PARP inhibitors

  • Drug sensitivity analysis can reveal associations between SFXN4 expression and response to specific therapeutics

These methodological considerations will help establish whether SFXN4 has value as a diagnostic, prognostic, or predictive biomarker in specific cancer types.

How do different fixation methods affect SFXN4 antibody performance in microscopy applications?

Fixation methods significantly impact SFXN4 antibody performance in immunofluorescence and immunohistochemistry applications:

• Paraformaldehyde (PFA) fixation:

  • Standard method: 4% PFA for 10-15 minutes at room temperature

  • Preserves mitochondrial morphology well

  • Maintains SFXN4 epitope accessibility for most antibodies

  • Recommended for co-localization studies with other mitochondrial markers

• Methanol fixation:

  • Cold methanol (-20°C) for 10 minutes

  • Can extract lipids and may alter membrane protein epitopes

  • Often increases accessibility to some internal epitopes

  • Test if PFA fixation gives weak signals for your specific SFXN4 antibody

• Glutaraldehyde:

  • Low concentrations (0.1-0.5%) in combination with PFA

  • Better preserves ultrastructure but may reduce epitope accessibility

  • May require more stringent permeabilization or antigen retrieval

• For tissue sections:

  • FFPE (formalin-fixed paraffin-embedded) tissues require antigen retrieval

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is commonly effective

  • Optimal antibody dilution for IHC: 1:20-1:200

• Permeabilization considerations:

  • For PFA-fixed cells, 0.1-0.2% Triton X-100 is typically sufficient

  • Digitonin (10-50 μg/ml) offers selective permeabilization of the plasma membrane while preserving mitochondrial membranes

  • Saponin (0.1%) provides milder permeabilization for preserved mitochondrial structure

When establishing a new immunofluorescence protocol with SFXN4 antibodies, it is advisable to test multiple fixation methods in parallel to determine optimal conditions for your specific antibody and experimental system.

What are the challenges in detecting endogenous SFXN4 protein levels and how can they be overcome?

Researchers face several challenges when detecting endogenous SFXN4 protein:

• Relatively low abundance:

  • SFXN4 is not as abundantly expressed as some mitochondrial proteins

  • Solution: Mitochondrial isolation/enrichment prior to Western blotting

  • Solution: Extended exposure times during imaging (while monitoring background)

• Membrane protein properties:

  • As a mitochondrial membrane protein, SFXN4 can aggregate during sample preparation

  • Solution: Use appropriate detergents (digitonin, DDM, or CHAPS)

  • Solution: Avoid boiling samples; instead heat at 70°C for 10 minutes

• Multiple protein isoforms:

  • Human SFXN4 has 3 isoforms produced by alternative splicing

  • Solution: Select antibodies raised against conserved regions

  • Solution: Use positive controls with known isoform expression

• Cross-reactivity with other SFXN family members:

  • SFXN family proteins share sequence homology

  • Solution: Choose antibodies validated against SFXN4 knockout samples

  • Solution: Include SFXN1/2/3/5 overexpression controls to confirm specificity

• Post-translational modifications:

  • These may affect epitope recognition

  • Solution: Use multiple antibodies targeting different epitopes

  • Solution: Validate results with genetic approaches (knockout/knockdown)

• In complex samples (tissues/tumors):

  • Variable expression across tissue types

  • Solution: Optimize antigen retrieval protocols for IHC

  • Solution: Use appropriate positive control tissues known to express SFXN4

• Quantification challenges:

  • Normalizing mitochondrial protein expression can be complex

  • Solution: Use mitochondrial markers (e.g., VDAC, TOM20) as loading controls

  • Solution: Consider mitochondrial mass markers to account for mitochondrial content variations

Following these approaches will improve detection sensitivity and specificity for endogenous SFXN4 in various experimental systems.

How can SFXN4 knockdown models be validated using antibody-based methods?

Validating SFXN4 knockdown models is essential for studying its biological functions. Here are methodological approaches using antibody-based techniques:

• Western blot validation:

  • Primary method to confirm protein level reduction

  • Compare band intensity at the expected molecular weight (37-38kDa) between control and knockdown samples

  • Quantify by densitometry with normalization to loading controls

  • Multiple technical and biological replicates are recommended for statistical analysis

• Immunofluorescence confirmation:

  • Visual confirmation of reduced signal intensity in cellular compartments

  • Co-staining with mitochondrial markers helps confirm specific reduction in mitochondrial SFXN4 signal

  • Quantitative image analysis for objective measurement of signal reduction

• Selection of appropriate controls:

  • Include non-targeting siRNA/shRNA controls

  • For CRISPR-Cas9 knockout models, include both wild-type cells and non-targeting guide controls

  • Rescue experiments: re-express SFXN4 to restore phenotype and protein detection

• Validation of knockdown efficiency:

  • Establish time course of protein depletion (SFXN4 half-life considerations)

  • Multiple siRNA sequences targeting different regions should produce similar phenotypes

  • For stable knockdown, confirm long-term reduction after puromycin selection

• Functional validation techniques:

  • Assess expected consequences of SFXN4 depletion:

    • Changes in mitochondrial complex I assembly (BN-PAGE analysis)

    • Altered iron metabolism markers

    • Impact on cell proliferation and invasion in cancer models

• Documentation standards:

  • Report the specific siRNA/shRNA sequences (e.g., "siRNA-1 as described previously ")

  • Include relevant antibody information (catalog number, dilution, exposure time)

  • Full-length blot images with molecular weight markers