MRPL58 Antibody

What is MRPL58 and why is it significant in mitochondrial research?

MRPL58 (mitochondrial ribosomal protein L58) is an essential component of the human mitoribosome. It functions as a peptidyl-tRNA hydrolase within the mitochondrial large ribosomal subunit, acting as a codon-independent translation release factor that has lost stop codon specificity. This protein is critical for the termination of translation in mitochondria, particularly in cases of abortive elongation. MRPL58 hydrolyzes peptidyl-tRNAs that have been prematurely terminated, which enables the recycling of stalled mitochondrial ribosomes .

The significance of MRPL58 lies in its essential role in mitochondrial protein synthesis and cell viability. Knockout studies have demonstrated that MRPL58 deficiency results in apoptosis, decreased mitochondrial membrane potential and mass, and reduced cytochrome c oxidase activity . This makes MRPL58 an important target for studying mitochondrial translation dynamics and related disorders.

What types of MRPL58 antibodies are available for research applications?

Available MRPL58 antibodies include polyclonal antibodies developed against the human MRPL58 protein. Specifically, rabbit-derived polyclonal antibodies against recombinant Protein Epitope Signature Tag (PrEST) antigen sequences are commercially available . These antibodies have been validated for several experimental applications, particularly Western blotting (WB) and immunocytochemistry (ICC) .

When selecting an appropriate MRPL58 antibody, researchers should consider:

• Target species specificity (human MRPL58 shares approximately 73% identity with mouse and rat orthologs)

• Required applications (validated vs. unvalidated uses)

• Clonality (polyclonal vs. monoclonal)

• Host species (to avoid cross-reactivity in multi-labeling experiments)

• Storage requirements (-20°C for long-term; +4°C for short-term storage)

What are the optimal storage conditions for maintaining MRPL58 antibody activity?

For optimal preservation of MRPL58 antibody activity, proper storage conditions are essential. The recommended storage protocol includes:

Antibody degradation typically occurs through repeated freeze-thaw cycles, prolonged storage at inappropriate temperatures, or contamination. To maintain antibody efficacy:

• Upon receipt, aliquot antibodies into smaller volumes based on typical experimental needs

• Store aliquots at -20°C for long-term preservation

• When using, thaw only the required aliquot at +4°C

• Return unused portion to +4°C if planning to use within 1-2 weeks; otherwise, avoid refreezing

Proper storage ensures consistent experimental results and extends the usable lifetime of the antibody preparation.

What are the validated applications for MRPL58 antibodies in cellular research?

MRPL58 antibodies have been validated for specific research applications, with Western blotting (WB) and immunocytochemistry (ICC) being the primary validated methods . Each application requires specific optimization:

Western Blotting (WB):

• Recommended dilution: 1:500-1:1000 (optimization required for specific antibody lots)

• Expected molecular weight: ~23 kDa

• Sample preparation: Total cell lysates or mitochondrial fraction enrichment

• Loading control recommendations: Use mitochondrial markers such as VDAC or COX IV for normalization

Immunocytochemistry (ICC):

• Recommended dilution: 1:100-1:500

• Fixation method: 4% paraformaldehyde followed by permeabilization

• Expected pattern: Punctate cytoplasmic staining consistent with mitochondrial localization

• Co-staining: Can be combined with mitochondrial markers like MitoTracker for confirmation

Additional applications may include immunoprecipitation for protein-protein interaction studies, though each new application requires validation by the researcher .

How can MRPL58 antibodies be delivered effectively into live cells for real-time mitochondrial studies?

For live-cell studies of MRPL58 function, researchers face the challenge of delivering antibodies across cell membranes. A promising "mix-and-go" strategy using cell-permeant bioadaptors has been developed for the cytosolic delivery of native antibodies to live mammalian cells . This approach offers several advantages for MRPL58 research:

• It requires no prior genetic or chemical modifications of commercially available antibodies

• The protocol operates under aqueous conditions at neutral pH

• It allows for immediate bioavailability of the delivered antibody

• It demonstrates minimal cytotoxicity compared to other delivery methods

The methodology involves:

• Preparing cell-permeant bioadaptors (like CpA1, CpA2, or CpT) that bind to the Fc domain of antibodies

• Simple mixing of the bioadaptor with the MRPL58 antibody

• Direct application to cell cultures

• GSH-triggered release of the antibody in the cytosol

• Target engagement of the delivered antibody with endogenous MRPL58

What controls should be included when using MRPL58 antibodies in experimental workflows?

Proper controls are essential for ensuring reliable and interpretable results when using MRPL58 antibodies. A comprehensive control strategy should include:

Positive Controls:

• Cell lines with confirmed MRPL58 expression (e.g., HeLa cells)

• Recombinant MRPL58 protein (when available)

• Tissues with known high mitochondrial content (heart, liver)

Negative Controls:

• MRPL58 knockout or knockdown samples (using CRISPR/Cas9 or siRNA)

• Isotype control antibodies (matching host species but non-targeting)

• Pre-absorption of antibody with immunizing peptide

Procedural Controls:

• Secondary antibody-only control (to assess non-specific binding)

• Loading controls for Western blot (mitochondrial and general proteins)

• Staining controls for immunocytochemistry (cytosolic and nuclear markers)

Proper implementation of these controls helps distinguish genuine MRPL58 signals from artifacts and enables confident interpretation of experimental results .

How can researchers address cross-reactivity issues when using MRPL58 antibodies?

Cross-reactivity can significantly impact experimental outcomes when using MRPL58 antibodies. The following strategies can help identify and mitigate such issues:

Identification of Cross-Reactivity:

• Western blot analysis revealing unexpected bands

• Staining patterns that don't match known MRPL58 subcellular localization

• Signal in tissues/cells known to lack or have minimal MRPL58 expression

Mitigation Strategies:

• Antibody Dilution Optimization: Test a range of dilutions to identify conditions that maximize specific binding while minimizing non-specific interactions.

• Blocking Optimization: Experiment with different blocking agents (BSA, milk, serum) and concentrations.

• Pre-absorption Control: Pre-incubate antibody with excess immunizing peptide to confirm specificity.

• Validation in MRPL58-depleted Samples: Use CRISPR-knockout or siRNA-knockdown samples as negative controls.

Cross-Species Applications:
When using human MRPL58 antibodies in other species, consider the sequence homology. For example, human MRPL58 shares approximately 73% sequence identity with both mouse and rat proteins . This moderate homology may allow cross-species reactivity but requires careful validation.

What are the common pitfalls in Western blot and ICC experiments with MRPL58 antibodies?

Researchers frequently encounter technical challenges when using MRPL58 antibodies. Understanding common pitfalls and their solutions enables more reliable experimental outcomes:

Western Blot Challenges:

Immunocytochemistry Challenges:

Technical Recommendations:

• Sample preparation is critical—use fresh samples with appropriate protease inhibitors

• For mitochondrial proteins like MRPL58, consider subcellular fractionation to enrich target

• Optimize fixation and permeabilization for ICC to preserve mitochondrial morphology

• Consider dual labeling with established mitochondrial markers to confirm localization

How can researchers validate the specificity of MRPL58 antibody signals in their experimental systems?

Validating antibody specificity is essential for generating reliable scientific data. For MRPL58 antibodies, a multi-faceted validation approach is recommended:

Genetic Validation:

• CRISPR/Cas9 Knockout: Generate MRPL58-null cell lines as negative controls (note that complete knockout may affect cell viability due to MRPL58's essential function)

• siRNA or shRNA Knockdown: Create transient or stable MRPL58-depleted cells and confirm signal reduction

• Overexpression: Transfect cells with MRPL58 expression constructs and verify signal increase

Biochemical Validation:

• Peptide Competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Immunoprecipitation-Mass Spectrometry (IP-MS): Confirm that MRPL58 is the primary protein captured by the antibody

• Orthogonal Antibodies: Use multiple antibodies targeting different epitopes of MRPL58

Functional Validation:

• Subcellular Localization: Confirm mitochondrial localization using co-staining with established markers

• Size Verification: Ensure detected band matches the predicted molecular weight (~23 kDa)

• Expression Pattern: Verify higher expression in tissues with abundant mitochondria

How can MRPL58 antibodies be used to investigate mitochondrial translation defects in disease models?

MRPL58 antibodies offer powerful tools for studying mitochondrial translation defects implicated in various diseases. Advanced research applications include:

Investigating Mitochondrial Disease Mechanisms:

• Protein-Protein Interaction Studies: Immunoprecipitation with MRPL58 antibodies can reveal interactions with other mitoribosome components or translation factors

• Mitoribosome Assembly Analysis: Using MRPL58 antibodies to track mitoribosome assembly defects in patient-derived cells

• Translation Termination Studies: Investigating MRPL58's role in rescuing stalled mitoribosomes in disease contexts

Disease Model Applications:
Research shows that MRPL58 (ICT1) gene knockout results in serious mitochondrial defects including:

• Apoptosis

• Decreased mitochondrial membrane potential

• Reduced mitochondrial mass

• Decreased cytochrome c oxidase activity

These phenotypes parallel certain mitochondrial diseases, making MRPL58 antibodies valuable for investigating:

• Mitochondrial encephalomyopathies

• Oxidative phosphorylation disorders

• Neurodegenerative diseases with mitochondrial involvement

Experimental Design Strategy for Disease Models:

• Compare MRPL58 levels and localization between patient-derived and control cells

• Assess correlation between MRPL58 function and mitochondrial translation efficiency

• Examine potential compensatory mechanisms through related factors like MTRFR, which plays a similar role in rescuing stalled mitoribosomes

• Investigate MRPL58 post-translational modifications that may be altered in disease states

What are the considerations for using MRPL58 antibodies in multi-protein complex analysis of mitochondrial ribosomes?

MRPL58 functions within the complex architecture of the mitochondrial ribosome, making antibodies against this protein valuable for studying multi-protein assemblies. Key considerations include:

Experimental Approaches for Complex Analysis:

• Co-Immunoprecipitation (Co-IP):

  • Optimize lysis conditions to maintain mitoribosome integrity

  • Use mild detergents (e.g., 0.5-1% Digitonin or 0.5% NP-40)

  • Consider crosslinking to capture transient interactions

  • Analyze precipitates for other mitoribosomal proteins and associated factors

• Proximity Labeling:

  • Engineering MRPL58 fusions with BioID or APEX2

  • Identifying neighboring proteins in the mitoribosomal environment

  • Comparing interactome differences between normal and pathological states

• Structural Analysis Integration:

  • Use antibodies as references in cryo-EM studies of mitoribosome structure

  • Compare experimental findings with published structural data on mitoribosome components

Technical Considerations:
Research has revealed that the interaction between subunits in mitochondria is not as extensive as in bacteria, with many bridges formed by mitochondria-specific RNA and protein components. The reduced peripheral contacts may result in increased conformational flexibility of mitoribosomal subunits . These structural insights suggest:

• Sample preparation must preserve native mitoribosome conformation

• Detergent choice and concentration are critical for maintaining complex integrity

• Buffer conditions should mimic the mitochondrial environment

How can MRPL58 antibodies contribute to understanding the differential roles of MRPL58 and related peptidyl-tRNA hydrolases like MTRFR?

MRPL58 belongs to a family of peptidyl-tRNA hydrolases that includes MTRFR, which plays a similar role in rescuing stalled mitoribosomes. Advanced research applications using MRPL58 antibodies can help delineate their distinct and overlapping functions:

Comparative Analysis Approaches:

• Functional Redundancy Studies:

  • Selective knockdown of either MRPL58 or MTRFR followed by antibody-based detection of the remaining protein

  • Assessment of compensatory upregulation

  • Mitochondrial translation efficiency measurements under single vs. double depletion

• Substrate Specificity Investigation:

  • Immunoprecipitation of MRPL58 vs. MTRFR to identify differentially associated tRNAs or nascent peptides

  • RNA-protein crosslinking followed by antibody-based purification

  • Mass spectrometry analysis of bound substrates

• Localization and Dynamics:

  • Super-resolution microscopy using differentially labeled antibodies against MRPL58 and MTRFR

  • Live-cell imaging using the cell-permeant bioadaptor delivery system

  • Quantitative analysis of co-localization under various cellular stress conditions

Biological Significance:
Research has shown that loss of MTRFR gene function causes mitochondrial translation defects, leading to encephalomyopathy, while MRPL58 is essential for cell viability . Understanding their differential roles has implications for:

• Developing targeted therapies for mitochondrial translation disorders

• Identifying compensatory mechanisms that could be therapeutically enhanced

• Understanding the evolution of translation termination mechanisms in mitochondria

How might advanced delivery methods enhance the utility of MRPL58 antibodies in mitochondrial research?

Emerging antibody delivery technologies offer exciting possibilities for MRPL58 research beyond traditional applications. The cell-permeant bioadaptor approach represents a significant advancement in this field:

Advanced Delivery Technologies:

The "mix-and-go" strategy using cell-permeant bioadaptors (CpA1, CpA2, and CpT) offers several advantages:

• No need for antibody modification

• Operation under physiological conditions

• Immediate bioavailability of delivered antibodies

• Lower cytotoxicity compared to electroporation or lipid-based methods

Future Applications for MRPL58 Research:

• Live-Cell Mitochondrial Translation Monitoring:

  • Delivery of fluorescently-labeled MRPL58 antibodies for real-time visualization

  • Tracking mitoribosome dynamics during translation cycles

  • Observing responses to stress conditions or drug treatments

• Targeted Protein Degradation:

  • Combining antibody delivery with "trim-away" technology for selective MRPL58 degradation

  • Using the TRIM21-derived cell-permeant bioadaptor (CpT) to achieve proteasome-based target degradation

  • Studying acute effects of MRPL58 depletion without genetic manipulation

• Therapeutic Development:

  • Potential for delivering function-blocking antibodies against mutant MRPL58

  • Testing mitochondrial translation modulators in patient-derived cells

  • Developing targeted approaches for mitochondrial diseases

This technology achieves delivery efficiency comparable to electroporation (approximately 95% positive cells) while offering advantages in throughput and cell viability .

What are emerging methodologies for studying MRPL58's role in mitochondrial quality control and stress responses?

As research on mitochondrial translation expands, new methodologies are emerging to study MRPL58's role in quality control and stress responses:

Innovative Methodological Approaches:

• Mitoribosome Profiling:

  • Adapting ribosome profiling techniques specifically for mitoribosomes

  • Using MRPL58 antibodies to selectively isolate translating mitoribosomes

  • Mapping translation pauses and termination events at single-nucleotide resolution

• Organelle-Specific Proximity Labeling:

  • Combining mitochondrial targeting with engineered MRPL58 variants

  • Identifying quality control factors that interact with MRPL58 during stress

  • Comparing interactomes under normal vs. oxidative stress conditions

• Multi-omics Integration:

  • Correlating MRPL58 protein levels/modifications with mitochondrial proteomics and transcriptomics

  • Developing predictive models of mitochondrial translation efficiency

  • Identifying biomarkers for mitochondrial dysfunction

Research Implications:
Studies have shown that MRPL58 knockout results in decreased mitochondrial membrane potential and cytochrome c oxidase activity . These findings suggest MRPL58 plays a key role in maintaining mitochondrial function during stress, with potential implications for:

• Aging research and intervention strategies

• Neurodegenerative disease mechanisms

• Cancer metabolism and therapeutic targeting

How can quantitative approaches be applied to MRPL58 antibody-based assays for translational research?

Quantitative analysis of MRPL58 using antibody-based assays offers significant potential for translational research applications:

Quantitative Methodologies:

• Multiplexed Antibody Assays:

  • Simultaneous detection of MRPL58 alongside other mitochondrial markers

  • Correlation with mitochondrial function parameters

  • Development of "mitochondrial health indices" for patient samples

• High-Content Imaging Analysis:

  • Automated quantification of MRPL58 levels and localization patterns

  • Machine learning approaches to identify subtle phenotypic changes

  • Correlation with clinical outcomes in patient-derived samples

• Absolute Quantification Strategies:

  • Developing quantitative Western blot protocols with recombinant standards

  • Mass spectrometry-based absolute quantification using antibody enrichment

  • Standardization across laboratories for clinical application

Translational Applications:

Research on related mitochondrial translation factors has already demonstrated biomarker potential. For example, ribosomal protein S18 (RPS18) and ribosome recycling factor (RRF) have been identified as high-confidence biomarkers for early Parkinson's disease . Similarly, MRPL58 quantification could provide insights into:

• Mitochondrial disease progression and treatment response

• Neurodegenerative disease risk assessment

• Cancer prognosis and therapy selection

Integrating these quantitative approaches with clinical data could establish MRPL58 as a valuable biomarker for mitochondrial dysfunction across multiple disease contexts.