Frataxin Antibody

What are the different forms of frataxin protein that can be detected with antibodies?

Frataxin exists in multiple forms that researchers must distinguish between when selecting appropriate antibodies. These include the cytosolic full-length frataxin (1-210) with a calculated molecular weight of 23 kDa, the mature mitochondrial frataxin (81-210) with an observed molecular weight of approximately 14 kDa, and intermediate forms such as mouse intermediate frataxin (41-207) . Different antibodies exhibit varying specificities for these forms, which influences experimental design and data interpretation. Researchers should verify which frataxin form their antibody detects, as some commercial antibodies fail to detect all forms or may detect non-specific proteins that run at similar molecular weights on PAGE .

What validation parameters should be assessed before using frataxin antibodies in experimental research?

Proper validation of frataxin antibodies requires systematic assessment across multiple parameters:

A comprehensive validation approach revealed that only one of five tested commercial antibodies successfully detected all frataxin standards while correctly identifying endogenous mature frataxin in mouse heart tissue . This underscores the critical importance of thorough validation before experimental use.

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

Successful Western blot detection of frataxin requires careful optimization of multiple parameters:

For frataxin detection, nitrocellulose membranes with 0.2 μm pore size have shown optimal results. Blocking should be performed with 5% milk in Dulbecco's phosphate-buffered saline containing 0.1% Tween-20 (DPBST) for 1 hour at room temperature . Primary antibody dilutions vary significantly between products, with recommended ranges from 1:500 for antibodies like Ab113691 and Ab175402 to 1:1000-1:6000 for others like 14147-1-AP . Overnight incubation at 4°C is typically required, followed by thorough washing in DPBST before applying secondary antibodies (anti-rabbit or anti-mouse HRP at 1:5000 dilution) . Researchers should note that sensitivity varies dramatically between different antibodies, necessitating optimization for each experimental system.

How do different detection methods for frataxin protein compare in terms of sensitivity and specificity?

Multiple methodologies have been developed for measuring frataxin levels, each with distinct advantages:

The choice of method depends on research requirements. For definitive identification of specific frataxin forms, mass spectrometry-based approaches using stable isotope dilution immunopurification with two-dimensional nano-ultrahigh performance liquid chromatography offer superior specificity and accuracy .

How can antibody specificity issues be addressed when studying frataxin across different species?

A critical consideration in frataxin research is the cross-species applicability of antibodies. Some antibodies like 14147-1-AP demonstrate reactivity with human, mouse, and rat samples, while others show species-specific detection patterns . When comparing frataxin across species, researchers should:

• Select antibodies raised against conserved regions of frataxin

• Validate each antibody with recombinant protein standards from each species

• Compare the observed molecular weights with predicted values (e.g., human mature frataxin at 15.3 kDa vs. mouse mature frataxin at 15.4 kDa)

• Consider using multiple antibodies targeting different epitopes to confirm findings

• Include appropriate positive controls from each species being studied

How can frataxin antibodies be utilized to study protein-protein interactions in the iron-sulfur cluster assembly pathway?

Frataxin functions as a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly, making antibody-based interaction studies valuable for understanding its mechanism:

Co-immunoprecipitation (CoIP) using frataxin antibodies can identify interaction partners within this pathway . When designing such experiments, researchers should select antibodies that do not interfere with binding interfaces. The search results indicate that specific antibodies have been validated for CoIP applications, suggesting their epitopes do not disrupt key protein-protein interaction surfaces .

Recent research has explored creating synthetic proteins (affitins) with high affinity for frataxin to modulate its interaction with the supercomplex. These molecular tools can serve as experimental probes to study how protein-protein interactions affect supercomplex activity . This approach, termed "protein function stabilization by quaternary addition," represents an innovative strategy for both studying frataxin's functional interactions and potentially developing therapeutic interventions.

What methodological approaches exist for using frataxin antibodies to evaluate therapeutic interventions for Friedreich's Ataxia?

Friedreich's Ataxia (FRDA) research focuses on upregulating or replacing frataxin protein, making antibody-based detection critical for evaluating therapeutic efficacy:

Preclinical studies in mouse and monkey models, as well as human cell lines, require well-validated antibodies to determine whether experimental approaches have successfully increased various forms of frataxin . Multiple antibody-based assays have been developed specifically for this purpose, including enzyme-linked immunosorbent assays that utilize mouse anti-frataxin antibodies for solid-phase immobilization and rabbit anti-frataxin antibodies as secondary detection reagents .

For therapeutic evaluation, researchers must consider:

• Whether the therapy increases the specific form of frataxin relevant to disease pathology

• How to distinguish between endogenous and therapeutically delivered frataxin

• The sensitivity required to detect potentially small increases in frataxin levels

• The need for quantitative rather than merely qualitative assessments

What strategies can resolve discrepancies in frataxin detection between different antibody-based methods?

Research has revealed significant discrepancies between antibodies in frataxin detection:

A systematic evaluation of five commercial antibodies found that three detected a protein in mouse heart tissue running at 23.4 kDa (near the expected size of full-length frataxin at 23.9 kDa), while only one antibody correctly detected all frataxin standards and endogenous mature frataxin without cross-reactivity to the 23.4 kDa protein . Such discrepancies highlight the need for comprehensive validation strategies.

When facing conflicting results, researchers should:

• Compare antibody immunogens and epitopes (e.g., antibodies raised against full-length human frataxin versus peptide fragments)

• Implement orthogonal detection methods, particularly mass spectrometry-based approaches

• Utilize genetic models (knockout/knockdown) as definitive negative controls

• Consider that different antibodies may recognize distinct conformational states or post-translationally modified forms of frataxin

How should molecular weight discrepancies be interpreted when analyzing frataxin Western blots?

Interpreting molecular weight variations requires understanding frataxin's processing and potential modifications:

The calculated molecular weight of full-length frataxin is 23 kDa, yet the mature form is observed at approximately 14 kDa following mitochondrial processing . Research has identified cases where antibodies detect proteins running at 23.4 kDa in mouse heart tissue, which is slightly faster than expected for full-length frataxin (23.9 kDa) . These discrepancies may reflect:

• Post-translational modifications altering migration patterns

• Alternative splicing or processing of frataxin

• Cross-reactivity with non-frataxin proteins

• Differences in electrophoresis conditions affecting protein migration

To address these challenges, researchers should include well-characterized recombinant protein standards of known molecular weight and use multiple antibodies targeting different epitopes to confirm identity .

What normalization strategies provide the most reliable quantification of frataxin levels in experimental samples?

Accurate quantification of frataxin requires appropriate normalization strategies:

The choice of normalization method should be consistent across experimental groups and validated for the specific research context. For absolute quantification, stable isotope dilution with mass spectrometry provides the highest accuracy .

How can novel antibody-based technologies enhance detection sensitivity for low-abundance frataxin in patient samples?

Advanced technologies are addressing the challenges of detecting low frataxin levels in FRDA patients:

Electrochemiluminescence assays (ECLIA) have demonstrated enhanced sensitivity for frataxin detection. These assays typically employ a primary mouse anti-frataxin monoclonal capture antibody (such as Chemicon Clone 1G2, #MAB1594) with a secondary rabbit polyclonal antibody for detection . The enhanced sensitivity makes these assays particularly valuable for detecting the reduced frataxin levels characteristic of FRDA.

Future developments may include:

• Single-molecule detection techniques for ultra-sensitive frataxin quantification

• Automated microfluidic platforms for high-throughput analysis

• Aptamer-based detection as alternatives to traditional antibodies

• Nanobody or single-domain antibody approaches for improved accessibility to conformational epitopes

What roles do frataxin antibodies play in developing novel therapeutic strategies like protein stabilization approaches?

Recent research has explored innovative therapeutic strategies involving frataxin-binding proteins:

The development of synthetic proteins (affitins) with high affinity for frataxin represents a novel approach termed "protein function stabilization by quaternary addition" . These small proteins can specifically bind frataxin and potentially:

• Alter frataxin's interaction with the supercomplex

• Modify the activity of the supercomplex

• Prevent degradation of unstable frataxin variants by acting as macromolecular tutors

• Modulate NFS1 (cysteine desulfurase) activity by altering enzyme dynamics

Antibodies play essential roles in evaluating these approaches, both in characterizing the interactions between frataxin and synthetic binding proteins and in assessing the functional consequences on supercomplex activity and frataxin stability .