IBA57 Antibody

What is IBA57 and why is it important to study with antibodies?

IBA57 is a nuclear-encoded mitochondrial protein that functions together with ISCA1 and ISCA2 in the reductive fusion of [2Fe-2S] clusters to [4Fe-4S] cofactors . The protein plays a crucial role in mitochondrial iron-sulfur cluster assembly, and defects in this process are associated with multiple mitochondrial dysfunction syndromes (MMDS) and neurological disorders.

When using antibodies to study IBA57, researchers can:

• Detect protein expression levels in different tissues and cell types

• Examine subcellular localization of IBA57

• Investigate protein-protein interactions within the iron-sulfur cluster assembly machinery

• Assess changes in IBA57 levels in disease states or experimental conditions

Methodologically, researchers should select antibodies validated against IBA57-depleted control samples (via RNAi or CRISPR knockout) to confirm specificity. Western blotting and immunofluorescence are primary applications, with optimization of mitochondrial fractionation techniques being essential for accurate detection.

What are the best experimental controls when using IBA57 antibodies?

Proper controls are essential for IBA57 antibody experiments:

• Positive controls: Include mitochondrial fractions from cells known to express IBA57 (HeLa cells are commonly used as demonstrated in published research)

• Negative controls:

  • RNAi-mediated depletion of IBA57 (60-80% reduction is typically achievable)

  • IBA57-knockout cell lines

  • Secondary antibody-only controls

• Rescue controls: Express RNAi-resistant wild-type IBA57 (smIBA57 wt) to confirm antibody specificity and restoration of signal

To verify antibody specificity, compare immunoblot signals between control, IBA57-depleted, and rescued samples, normalizing to mitochondrial markers or loading controls like β-actin as shown in published studies .

How can IBA57 antibodies be used to investigate iron-sulfur cluster assembly defects?

IBA57 antibodies can be instrumental in characterizing iron-sulfur (Fe-S) cluster assembly defects through a multi-faceted approach:

Methodological approach:

• Use IBA57 antibodies in immunoblotting to quantify protein levels in patient-derived cell lines or model systems

• Compare with protein levels of other Fe-S assembly components (particularly NFU1, which is affected by IBA57 deficiency)

• Correlate IBA57 levels with activities of Fe-S dependent enzymes

Research has shown that IBA57 deficiency impacts multiple [4Fe-4S] proteins, particularly:

• Mitochondrial aconitase (ACO2)

• Succinate dehydrogenase (SDH/Complex II)

• Lipoic acid synthetase (LIAS)

In experimental designs, researchers should incorporate measurement of these downstream targets alongside IBA57 detection. Published data shows that IBA57 depletion reduces SDH activity by more than 75% and aconitase activity by more than 30% .

What is the relationship between IBA57 and NFU1, and how can antibodies help elucidate this connection?

The relationship between IBA57 and NFU1 represents an important area for investigation in mitochondrial Fe-S cluster assembly research:

NFU1 and IBA57 functional relationship:

• NFU1 is a late-targeting factor in the ISC assembly machinery

• It is specifically required for assembly of a subset of [4Fe-4S] proteins to respiratory complexes I, II, and LIAS

• IBA57 deficiency leads to decreased NFU1 protein expression

Methodological approach with antibodies:

• Use both IBA57 and NFU1 antibodies in parallel western blots

• Perform IBA57 knockdown/knockout experiments and measure NFU1 levels

• Conduct co-immunoprecipitation with IBA57 antibodies to investigate direct or indirect interactions

• Employ proximity ligation assays to detect potential in situ interactions

Research has demonstrated that patients with IBA57 mutations show decreased NFU1 protein levels, suggesting that "IBA57 regulates the expression of NFU1, which subsequently affects the expression of lipoate-containing protein as well as respiratory complex II" .

How can IBA57 antibodies be used to study pathogenic variants and their effects?

IBA57 antibodies are valuable tools for investigating the functional consequences of disease-associated IBA57 variants:

Methodological approach:

• Express wild-type and mutant IBA57 in cell models

• Use antibodies to verify protein expression levels

• Compare cellular localization patterns

• Assess stability of mutant proteins over time

For example, in a study of hereditary necrotizing myelopathy (HNM) in Kooiker dogs, researchers identified an R147W mutation in IBA57 . They established a cell culture model using:

• RNAi-mediated depletion of endogenous IBA57

• Expression of RNAi-resistant wild-type or mutant (R147W) IBA57

• Validation using IBA57 antibodies for western blotting

The experimental results revealed that while wild-type IBA57 could restore mitochondrial [4Fe-4S] protein assembly, the R147W mutant was inefficient, particularly in restoring aconitase and SDH activities .

What are the optimal sample preparation methods for detecting IBA57 with antibodies?

Effective detection of IBA57 requires careful sample preparation due to its mitochondrial localization:

Methodological approach for mitochondrial fractionation:

• Use digitonin-based cell fractionation to obtain mitochondria-enriched fractions

• Optimize detergent concentration to preserve mitochondrial integrity

• Include protease inhibitors to prevent degradation

• Process samples rapidly and keep cold throughout

Whole cell lysate preparation:

• Use RIPA or similar buffers for comprehensive protein extraction

• Sonicate briefly to ensure complete lysis of mitochondria

• Centrifuge to remove cellular debris

• Normalize protein concentration before immunoblotting

Research studies typically use these approaches to detect IBA57 via western blotting with normalization to mitochondrial markers or β-actin .

How can IBA57 antibodies be combined with enzyme activity assays to comprehensively assess functional defects?

Combining IBA57 antibody detection with functional enzyme assays provides a powerful approach to correlate protein levels with functional outcomes:

Methodological integration:

• Perform immunoblotting with IBA57 antibodies on one portion of your samples

• Use parallel samples for spectrophotometric enzyme activity assays:

  • Aconitase activity assay

  • Succinate dehydrogenase (SDH) activity

  • Citrate synthase (as reference enzyme)

• Correlate IBA57 protein levels with enzyme activities

This approach has been validated in research where IBA57 depletion reduced SDH activity by >75% and aconitase activity by >30% without affecting citrate synthase .

The table below summarizes typical findings when comparing control, IBA57-depleted, and rescued cells:

These data illustrate the typical pattern observed in functional studies of IBA57 and its mutations .

How can IBA57 antibodies be used to study mitochondrial disorders in patient samples?

IBA57 antibodies are valuable tools for investigating IBA57-related mitochondrial disorders in patient-derived samples:

Methodological approach:

• Obtain patient fibroblasts, myoblasts, or tissue samples

• Process for immunoblotting with IBA57 antibodies

• Compare IBA57 protein levels to age-matched controls

• Correlate with clinical severity and genetic findings

• Analyze downstream markers of mitochondrial dysfunction

In patient studies, researchers have observed:

• Substantially decreased IBA57 protein in patient-derived myoblasts and fibroblasts

• Little to no detection of truncated IBA57 proteins from mutant alleles

• Downstream effects on respiratory complex activities

For clinical research, it's important to combine IBA57 detection with comprehensive assessment of mitochondrial function and patient phenotyping.

What are the considerations when using IBA57 antibodies in neurological disease research?

IBA57 mutations are associated with progressive cavitating leukoencephalopathy (PCL) and other neurological conditions, making IBA57 antibodies valuable in neuroscience research:

Methodological considerations:

• Brain tissue analysis requires optimized fixation for IBA57 immunohistochemistry

• Cell-type specific expression analysis may require co-staining with neuronal, glial, and mitochondrial markers

• Regional differences in brain IBA57 expression may correlate with selective vulnerability

Research has shown that IBA57 deficiency affects white matter integrity through disruption of mitochondrial energy metabolism . Patients with IBA57 mutations present with neuroimaging features similar to those with NFU1 mutations, including leukoencephalopathy in the periventricular white matter and corpus callosum, with partial cystic degeneration or cavitation .

When studying these conditions, researchers should correlate IBA57 antibody staining patterns with neuroimaging findings and clinical manifestations to advance understanding of disease mechanisms.

What are common issues when working with IBA57 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with IBA57 antibodies:

Non-specific bands in western blots:

• Increase blocking time and concentration

• Optimize primary antibody dilution (typically 1:500 to 1:2000)

• Include positive and negative controls (IBA57-depleted samples)

• Verify with multiple antibodies targeting different epitopes

Low signal intensity:

• Enrich for mitochondrial fractions

• Increase protein loading (30-50 μg for whole cell lysates)

• Optimize exposure time

• Use enhanced chemiluminescence detection systems

• Consider signal amplification methods

Inconsistent results between experiments:

• Standardize cell culture conditions (density, passage number)

• Use internal loading controls consistently

• Maintain strict temperature control during sample preparation

• Prepare fresh samples when possible

Published protocols have demonstrated successful IBA57 detection using standard immunoblotting techniques with appropriate optimization .