msto1 Antibody

What are the validated applications for MSTO1 antibodies?

MSTO1 antibodies have been validated for multiple research applications, with the most commonly confirmed uses being Western Blotting (WB), Immunohistochemistry on paraffin-embedded sections (IHC-P), and in some cases, ELISA. According to multiple antibody manufacturers, the recommended dilutions typically range from 1:500-1:2000 for WB and IHC applications . When planning experiments, it's essential to validate the antibody in your specific experimental conditions as reactivity can vary significantly between sample types and applications.

What is the subcellular localization of MSTO1 and how does this impact antibody selection?

The subcellular localization of MSTO1 has been a subject of scientific debate. While initially reported to be mitochondrial, more recent studies have shown that MSTO1 is primarily a cytoplasmic protein with limited colocalization with mitochondria (approximately 30% colocalization in intact cells) . Immunoblotting experiments on cell subfractions revealed that MSTO1 is predominantly found in the cytosolic fraction (100,000g supernatant) and not in the mitochondrial pellet . When selecting antibodies, researchers should consider this predominantly cytoplasmic localization and potentially use cytoplasmic extraction protocols rather than mitochondrial enrichment methods for optimal detection.

What is the molecular weight of MSTO1 protein and what bands should I expect on Western blots?

The observed molecular weight of MSTO1 protein on SDS-PAGE is typically between 57-62 kDa . This is consistent with the predicted size based on the amino acid sequence. When performing Western blot analysis, a single band within this range should be observed in human samples. If using recombinant tagged MSTO1 proteins (such as HA-tagged or Myc-tagged MSTO1), the molecular weight will be slightly higher depending on the tag size.

How should I optimize Western blotting protocols for MSTO1 detection?

For optimal Western blotting results with MSTO1 antibodies:

• Sample preparation: Total cell lysates from human cell lines (HeLa, HepG2) have been successfully used for MSTO1 detection . For subcellular studies, cytoplasmic fractions rather than mitochondrial fractions yield stronger signals due to MSTO1's predominantly cytoplasmic localization .

• Loading amount: Load 25-30 μg protein per lane for standard cell lysates.

• Gel percentage: Use 7.5% SDS-PAGE for better resolution of MSTO1.

• Transfer conditions: Standard semi-dry or wet transfer protocols are suitable.

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody dilution: Start with 1:1000 dilution and adjust as needed based on signal intensity.

• Incubation conditions: Overnight at 4°C for primary antibody provides optimal results.

Research has shown that MSTO1 protein abundance may be significantly decreased in cells carrying MSTO1 mutations (to as low as 15% of normal levels in some cases) , so optimization of detection protocols is crucial for mutation research.

What are the considerations for immunohistochemical detection of MSTO1?

For successful immunohistochemical detection of MSTO1:

• Antigen retrieval: Manufacturers recommend TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative . Optimization of antigen retrieval is critical for specific staining.

• Tissue specificity: MSTO1 is ubiquitously expressed, but antibody validation has been specifically reported in human ovary cancer tissue .

• Dilution range: Begin with 1:500-1:2000 dilution and titrate as needed.

• Controls: Include both positive (HeLa or HepG2 cells) and negative controls (omission of primary antibody).

• Detection system: Standard HRP/DAB detection systems are suitable for MSTO1 detection.

When interpreting results, remember that MSTO1 displays primarily cytoplasmic staining patterns with some studies showing partial mitochondrial colocalization .

How can I validate the specificity of MSTO1 antibodies in my experiments?

Validating antibody specificity is crucial for reliable results. For MSTO1 antibodies, consider these validation approaches:

• Knockdown/knockout controls: Use siRNA or CRISPR to generate MSTO1-depleted cells. Studies have shown that RNAi-mediated MSTO1 knockdown causes mitochondrial fragmentation , which can serve as a phenotypic confirmation of successful knockdown.

• Overexpression controls: Overexpression of recombinant MSTO1 (wild-type or tagged versions) provides positive controls. Research has demonstrated that overexpression of MSTO1 can rescue mitochondrial fusion dynamics in patient fibroblasts .

• Patient-derived cells: Fibroblasts from patients with MSTO1 mutations show drastically reduced MSTO1 protein levels (approximately 15% of normal) , providing another validation approach.

• Peptide competition: Pre-incubation of the antibody with the immunogenic peptide should abolish specific signals.

• Multiple antibodies: Use antibodies raised against different epitopes of MSTO1 to confirm consistent detection patterns.

How do mutations in MSTO1 affect protein detection with antibodies?

Research on MSTO1 mutations has revealed important considerations for antibody detection:

When studying patient samples with MSTO1 mutations, researchers should be aware that the protein may be present at very low levels or completely absent. This necessitates highly sensitive detection methods and appropriate loading controls. qPCR analysis of MSTO1 mRNA can provide complementary information, as patient fibroblasts have shown 36-42% of normal MSTO1 mRNA expression .

How can MSTO1 antibodies be used to study mitochondrial dynamics?

MSTO1 antibodies can be valuable tools for investigating mitochondrial dynamics:

• Co-immunoprecipitation studies: MSTO1 interacts with the mitochondrial fusion machinery . Antibodies can be used to pull down MSTO1 and identify interaction partners.

• Immunofluorescence microscopy: While some studies report limitations in using anti-MSTO1 antibodies for immunofluorescence , tagged versions (MSTO1-HA, MSTO1-Myc) have been successfully used to study localization patterns. These studies revealed approximately 30% colocalization with mitochondria in intact cells, increasing to around 50% after plasma membrane permeabilization .

• Protein release assays: MSTO1 antibodies have been used in protein release assays following plasma membrane permeabilization, revealing that MSTO1 is promptly released into the cytoplasmic fraction similar to other soluble cytosolic proteins like α-tubulin .

• Rescue experiments: Antibodies can verify successful expression of wild-type MSTO1 in rescue experiments, where overexpression of MSTO1 restored normal mitochondrial fusion dynamics in patient fibroblasts .

When designing such experiments, consider that MSTO1 contains 2 tubulin/Ftz-like GTPase domains with predicted GTP binding sites , which may influence protein-protein interactions.

What do we know about MSTO1's role in pathophysiological conditions based on antibody studies?

Antibody-based studies have revealed several key insights into MSTO1's role in pathophysiology:

• Mitochondrial fragmentation: In patient-derived fibroblasts with MSTO1 mutations, antibody studies revealed drastically reduced MSTO1 protein levels correlating with mitochondrial fragmentation, aggregation, and decreased network continuity .

• Fusion defects: MSTO1-deficient cells show impaired mitochondrial fusion that can be rescued by MSTO1 overexpression, as confirmed by antibody detection .

• mtDNA depletion: Cells expressing MSTO1 mutations show decreased mtDNA copy number compared to wild-type (demonstrated through in vitro experiments with tagged mutant proteins) .

• Disease mechanism: Antibody-based studies helped establish that MSTO1 is a cytoplasmic pro-mitochondrial fusion protein rather than an integral mitochondrial protein , changing our understanding of its mechanism of action.

These findings have established MSTO1 mutations as a cause of rare mitochondrial disorders characterized primarily by myopathy and cerebellar ataxia, but also involving multisystem features in some patients .

Why might I be unable to detect MSTO1 in some tissue samples?

Researchers have reported challenges in detecting MSTO1 in certain sample types. For example, one study noted no immunoreactive signal at the expected molecular weight even in control muscle homogenates . Consider these possible explanations and solutions:

• Tissue-specific expression levels: While MSTO1 is ubiquitously expressed, levels may vary across tissues.

• Extraction methods: Some lysis buffers may not effectively solubilize MSTO1. Consider using RIPA buffer with protease inhibitors.

• Protein degradation: MSTO1 may be susceptible to degradation in certain sample types. Use fresh samples and maintain cold conditions throughout processing.

• Antibody limitations: Some antibodies may have tissue-specific limitations. Try alternative antibodies targeting different epitopes.

• Detection sensitivity: Consider using more sensitive detection methods like chemiluminescent substrates with longer exposure times.

How can I differentiate between wild-type and mutant MSTO1 using antibodies?

Differentiating wild-type from mutant MSTO1 can be challenging but several approaches may help:

• Quantitative assessment: Most MSTO1 mutations result in reduced protein levels rather than size differences. Quantitative Western blotting with appropriate normalization can reveal these differences in expression levels.

• Epitope-specific antibodies: If the mutation affects an epitope recognized by a specific antibody, loss of signal with that particular antibody could indicate the mutation.

• Functional assays: Combine antibody detection with functional assays. For example, MSTO1 mutations impair mitochondrial fusion, which can be assessed using photoactivatable GFP (mtPA-GFP) alongside antibody detection of MSTO1 .

• Recombinant expression: Express tagged versions of wild-type and mutant MSTO1 to compare their stability and localization patterns using tag-specific antibodies .

• Mass spectrometry: For specific mutations, follow antibody-based purification with mass spectrometry analysis to identify the exact protein variant.

Why might MSTO1 antibody signal vary between cytoplasmic and mitochondrial fractions?

Studies have revealed that MSTO1 is predominantly cytoplasmic with limited mitochondrial localization . When fractionating cells, researchers should be aware of these distribution patterns:

• Predominant cytosolic localization: MSTO1 is primarily detected in the 100,000g (cytosolic) supernatant fraction after ultracentrifugation .

• Limited mitochondrial association: Only approximately 30% colocalization with mitochondrial markers in intact cells, increasing to around 50% after plasma membrane permeabilization .

• Protein release upon permeabilization: MSTO1 is promptly released upon plasma membrane permeabilization, indicating it's not an integral membrane protein .

• Extraction efficiency: When normalized to mitochondrial loading controls like prohibitin, MSTO1 protein amount was relatively low (only 35%) in mitochondrial fractions compared to total cell lysate .

For accurate assessment, use multiple fractionation controls: cytosolic markers (e.g., α-tubulin), mitochondrial markers (e.g., prohibitin), and markers for other cellular compartments to ensure clean fractionation.

How can MSTO1 antibodies be used to explore potential therapeutic targets?

MSTO1 antibodies can facilitate research into potential therapeutic approaches for MSTO1-related disorders:

• Target validation: Antibodies can confirm successful modulation of MSTO1 expression or function in drug screening assays.

• Rescue experiments: Studies have shown that overexpression of wild-type MSTO1 can rescue mitochondrial fusion defects in patient cells . Antibodies can verify protein expression in such therapeutic approaches.

• Interaction studies: Antibodies can help identify and validate proteins that interact with MSTO1, potentially revealing alternative therapeutic targets within the same pathway.

• Biomarker development: MSTO1 antibodies might help develop biomarkers for diagnosis or treatment monitoring in MSTO1-related disorders.

• Structural studies: Antibodies recognizing specific domains (tubulin domains or GTPase regions) could provide insights into functional regions for targeted drug development.

When designing such experiments, consider that studies have identified seven possible GTP binding sites in the first tubulin/Ftz GTPase domain of MSTO1 , which might be relevant for small molecule targeting strategies.

What technical advances might improve MSTO1 antibody applications in research?

Several technical advances could enhance MSTO1 antibody applications:

• Domain-specific antibodies: Development of antibodies targeting specific functional domains (tubulin domains, GTPase regions) could provide better tools for structure-function studies.

• Proximity labeling applications: Combining MSTO1 antibodies with proximity labeling techniques (BioID, APEX) could better characterize its transient interactions at the mitochondrial outer membrane.

• Live-cell compatible antibody fragments: Development of cell-permeable antibody fragments could enable live-cell imaging of endogenous MSTO1.

• Super-resolution microscopy compatibility: Validation of antibodies for super-resolution techniques could provide better insights into MSTO1's spatial relationship with mitochondria.

• Phospho-specific antibodies: If MSTO1 undergoes post-translational modifications regulating its function, phospho-specific antibodies could reveal regulatory mechanisms.

These technical advances would further our understanding of MSTO1's role in mitochondrial dynamics and potentially identify new therapeutic targets for MSTO1-related disorders.