MRPL50 Antibody

What is MRPL50 and why is it significant in mitochondrial research?

MRPL50 (also known as L50mt or MRP-L50) is a component of the 39S large subunit of the mitochondrial ribosome. This 18 kDa protein plays a critical role in mitochondrial protein synthesis, particularly for proteins involved in oxidative phosphorylation. Recent research has demonstrated that MRPL50 deficiency results in destabilization of the large mitochondrial ribosomal subunit while preserving the small subunit, leading to impaired mitochondrial translation .

MRPL50 has gained significant research interest following the discovery that homozygous missense variants in MRPL50 are associated with a syndrome characterized by primary ovarian insufficiency (POI), bilateral high-frequency sensorineural hearing loss, kidney dysfunction, and cardiac abnormalities . This clinical presentation highlights the critical importance of mitochondrial translation in high-energy-demand tissues and positions MRPL50 as an important research target for understanding mitochondrial biology and associated pathologies.

What applications are validated for MRPL50 antibodies?

MRPL50 antibodies have been validated for multiple research applications with specific recommended parameters:

It is recommended to titrate antibody concentrations in each experimental system to obtain optimal results, as sensitivity may be sample-dependent . For immunohistochemistry applications, antigen retrieval with TE buffer at pH 9.0 is suggested, with citrate buffer at pH 6.0 as an alternative .

What are the key technical specifications of available MRPL50 antibodies?

Commercially available MRPL50 antibodies possess the following characteristics:

When selecting an antibody for your research, consider the specific application requirements and validated reactivity to ensure optimal experimental outcomes.

How should I design experiments to study MRPL50's role in mitochondrial ribosome function?

To investigate MRPL50's role in mitochondrial ribosome function, consider the following experimental design approach:

• Expression manipulation studies:

  • Knockdown/knockout strategies using siRNA, shRNA, or CRISPR-Cas9

  • Rescue experiments with wild-type and mutant MRPL50

  • Inducible expression systems for temporal control

• Functional readouts:

  • Mitochondrial translation efficiency (pulse-labeling with 35S-methionine)

  • Respiratory chain complex abundance (particularly Complex I)

  • Oxygen consumption rates and ATP production

  • Mitochondrial membrane potential

• Structural analysis:

  • Mitochondrial ribosome integrity assessment via sucrose gradient centrifugation

  • Co-immunoprecipitation with other mitochondrial ribosomal proteins

  • Blue Native PAGE for analysis of assembled complexes

• Cellular models:

  • Established cell lines (HeLa, HepG2, HT-1080)

  • Patient-derived fibroblasts carrying MRPL50 mutations

  • Model organisms (Drosophila has been validated)

The experimental design should include appropriate controls, particularly when using antibody-based detection methods, and consider the tissue-specific effects observed in clinical cases of MRPL50 deficiency .

What is the optimal protocol for Western blot detection of MRPL50?

For optimal Western blot detection of MRPL50:

Sample Preparation:

• Prepare cell lysates in RIPA buffer supplemented with protease inhibitors

• For mitochondrial enrichment, consider differential centrifugation protocols

• Determine protein concentration using Bradford or BCA assay

• Load 20-50 μg total protein per lane

Gel Electrophoresis and Transfer:

• Use 12-15% SDS-PAGE gels (optimal for 18 kDa proteins)

• Include molecular weight markers spanning 10-25 kDa range

• Transfer to PVDF or nitrocellulose membranes (0.2 μm pore size recommended)

Antibody Incubation:

• Block membranes in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary MRPL50 antibody at 1:500-1:2000 dilution overnight at 4°C

• Wash 3x with TBST

• Incubate with HRP-conjugated secondary antibody (anti-rabbit) at 1:5000 dilution for 1 hour

• Wash 3x with TBST

Detection and Controls:

• Visualize using ECL substrate and appropriate imaging system

• Expected band size: 18 kDa

• Positive control: HepG2, HeLa, or HT-1080 cell lysates

• Loading control: β-actin or GAPDH for total lysates; VDAC or TOM20 for mitochondrial fractions

• Specificity control: MRPL50 knockdown/knockout samples if available

This protocol has been validated to detect endogenous MRPL50 in multiple human cell lines .

How can I optimize immunohistochemistry protocols for MRPL50 in tissue samples?

For successful MRPL50 immunohistochemistry in tissue samples:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin

• Section at 4-5 μm thickness

• Mount on positively charged slides

Antigen Retrieval (Critical Step):

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

• Heat-induced epitope retrieval: Pressure cooker or microwave heating

Staining Protocol:

• Block endogenous peroxidase (3% H₂O₂, 10 minutes)

• Protein block (5% normal goat serum, 1 hour)

• Primary antibody incubation: Anti-MRPL50 at 1:50-1:500 dilution

• Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: HRP-polymer and DAB chromogen

• Counterstain: Hematoxylin

Validation and Controls:

• Positive tissue control: Human liver tissue (validated for MRPL50 expression)

• Negative controls:

  • Primary antibody omission

  • Non-immune IgG at matching concentration

• Expected staining pattern: Punctate cytoplasmic staining consistent with mitochondrial localization

This protocol should be optimized for each tissue type, with special attention to antigen retrieval conditions and antibody dilution for optimal signal-to-noise ratio.

How should I interpret changes in MRPL50 expression in relation to mitochondrial dysfunction?

Interpreting MRPL50 expression changes requires careful consideration of several factors:

Expression Pattern Analysis:

• Decreased MRPL50 levels may indicate mitochondrial ribosome large subunit destabilization, as observed in patient fibroblasts with MRPL50 mutations

• Changes should be normalized to total mitochondrial content using markers like VDAC or TOM20

• Compare MRPL50 levels with other mitochondrial ribosomal proteins to distinguish specific versus general ribosomal effects

Functional Correlations:
Based on research findings, MRPL50 deficiency leads to specific downstream effects that should be assessed:

Causal Relationship Assessment:

• Determine if MRPL50 changes are primary (causative) or secondary (adaptive) to mitochondrial dysfunction

• Conduct time-course experiments following mitochondrial stress induction

• Perform rescue experiments with wild-type MRPL50 to confirm causality

When interpreting results, consider that the biochemical phenotype associated with MRPL50 variants includes specific destabilization of the large mitochondrial ribosomal subunit while preserving the small subunit, leading to oxidative phosphorylation deficiency with particular impact on Complex I .

What can co-localization studies reveal about MRPL50 function in mitochondria?

Co-localization studies provide valuable insights into MRPL50's spatial organization and functional interactions within mitochondria:

Expected Co-localization Patterns:

• MRPL50 should co-localize with markers of the mitochondrial matrix where mitochondrial ribosomes function

• Significant overlap with other mitochondrial ribosomal large subunit proteins (e.g., MRPL3, MRPL12, MRPL44)

• Partial co-localization with nucleoids (mtDNA-protein complexes) where translation occurs near transcription sites

• Potential dynamic association with inner mitochondrial membrane where OXPHOS complexes are assembled

Analytical Approaches:

• Quantitative co-localization metrics (Pearson's coefficient, Manders' overlap)

• Super-resolution microscopy for detailed spatial relationships

• Live-cell imaging with fluorescently tagged MRPL50 to observe dynamic associations

• Proximity ligation assay to detect specific protein-protein interactions in situ

Functional Interpretations:

• Altered co-localization patterns in pathological states may indicate:

  • Disrupted mitochondrial ribosome assembly

  • Impaired coupling between translation and OXPHOS complex assembly

  • Compensatory responses to mitochondrial stress

When analyzing co-localization data, consider that MRPL50 deficiency specifically affects Complex I biogenesis , suggesting potential spatiotemporal regulation of mitochondrial translation for different OXPHOS complexes that might be visible through advanced imaging techniques.

How does MRPL50 deficiency specifically impact Complex I compared to other respiratory chain complexes?

Research has revealed that MRPL50 deficiency has a particularly pronounced effect on Complex I of the respiratory chain . This specificity warrants detailed investigation:

Differential Impact Assessment:

• Quantitative proteomics and western blot analysis of patient fibroblasts demonstrate a mild but significant decrease in Complex I abundance while other complexes may be less affected

• This pattern differs from some other mitochondrial translation defects that more broadly impact multiple respiratory chain complexes

Mechanistic Hypotheses:

• Translation Efficiency Hypothesis: MRPL50 may play a specific role in translating mitochondrial mRNAs encoding Complex I subunits

• Assembly Coupling Hypothesis: MRPL50 might facilitate co-translational insertion of newly synthesized Complex I components

• Stability Regulation Hypothesis: MRPL50 deficiency could indirectly affect Complex I through altered quality control mechanisms

Experimental Approaches for Investigation:

• Comparative Analysis: Measure all respiratory chain complexes using:

  • Blue Native PAGE coupled with immunoblotting

  • Enzyme activity assays for each complex

  • Targeted proteomics of representative subunits

• Translation Kinetics: Pulse-chase labeling to compare synthesis rates of mitochondrial-encoded subunits across different complexes

• Assembly Intermediate Analysis: Identify potential accumulation of specific Complex I assembly intermediates in MRPL50-deficient cells

• Functional Impact: Assess:

  • NADH:ubiquinone oxidoreductase activity

  • ROS production

  • Membrane potential dependencies

This Complex I-specific effect provides an important clue about the specialized functions of mitochondrial ribosomal proteins beyond their structural roles in the mitoribosome .

How can I investigate the role of MRPL50 in mitochondrial disease pathogenesis?

MRPL50 deficiency has been linked to a syndromic disorder featuring primary ovarian insufficiency (POI), sensorineural hearing loss, kidney dysfunction, and cardiac abnormalities . To investigate its role in disease pathogenesis:

Genetic Analysis Approaches:

• Screen for MRPL50 variants in cohorts with:

  • Unexplained POI, especially with syndromic features

  • Mitochondrial disease presentations with tissue-specific manifestations

  • Familial hearing loss with multisystem involvement

• Variant characterization:

  • Assess conservation of affected residues across species

  • Perform in silico structural predictions

  • Evaluate variant frequency in population databases

Functional Validation Studies:

• Patient-derived cell models:

  • Fibroblast analysis has successfully demonstrated biochemical phenotypes

  • Consider generating induced pluripotent stem cells (iPSCs) and differentiation to affected tissue types

• CRISPR-engineered models:

  • Generate isogenic cell lines with specific MRPL50 variants

  • Create animal models (the Drosophila model has been validated for ovarian development studies)

Tissue-Specific Pathogenesis Investigation:
Based on clinical presentations, focus on mechanisms in:

Therapeutic Exploration:

• Test mitochondrial-targeted compounds (antioxidants, metabolic modifiers)

• Investigate approaches to stabilize mitochondrial ribosome assembly

• Explore gene therapy or gene editing strategies

This multifaceted approach can help elucidate how MRPL50 deficiency leads to tissue-specific pathology and identify potential therapeutic interventions .

What techniques can determine the structural role of MRPL50 in the mitochondrial ribosome?

Understanding MRPL50's structural role requires sophisticated biochemical and biophysical approaches:

Ribosome Assembly Analysis:

• Sucrose gradient ultracentrifugation:

  • Compare sedimentation profiles of mitochondrial ribosomal subunits between normal and MRPL50-deficient cells

  • Identify assembly intermediates that accumulate in the absence of MRPL50

  • Quantify the ratio of assembled versus unassembled large subunits

• Proximity labeling techniques:

  • BioID or APEX2 fused to MRPL50 to identify proximal proteins

  • Map the neighborhood of MRPL50 within the mitoribosome

• Crosslinking mass spectrometry:

  • Identify direct interaction partners of MRPL50

  • Map contact residues for structural modeling

Structural Biology Approaches:

• Cryo-electron microscopy:

  • Compare structures of normal and MRPL50-deficient mitoribosomes

  • Identify structural perturbations in the large subunit

  • Analyze potential effects on functional sites (peptidyl transferase center, exit tunnel)

• Hydrogen-deuterium exchange mass spectrometry:

  • Assess conformational changes in the large subunit upon MRPL50 depletion

  • Identify regions with altered solvent accessibility

Functional Structural Analysis:

• Structure-guided mutagenesis:

  • Generate point mutations in key MRPL50 residues

  • Assess effects on ribosome assembly and translation

  • Correlate with patient-derived mutations (e.g., p.Val112Asp)

• Domain mapping:

  • Create truncation constructs to identify essential regions

  • Test capacity of mutants to rescue MRPL50 deficiency phenotypes

These approaches would provide comprehensive insights into how MRPL50 contributes to mitochondrial ribosome structure and function, potentially explaining the specific downstream effects on Complex I biogenesis observed in patient cells .

How can differential proteomics be used to characterize MRPL50-associated mitochondrial translation defects?

Differential proteomics offers powerful approaches to characterize the molecular consequences of MRPL50 deficiency:

Sample Preparation Strategies:

• Cellular fractionation: Isolate highly purified mitochondria to focus on mitochondrial proteome changes

• Temporal analysis: Examine acute versus chronic MRPL50 depletion to distinguish primary effects from adaptive responses

• Tissue-specific profiling: Compare proteome alterations across different tissue types (if using animal models)

Quantitative Proteomics Approaches:

• Global proteomics:

  • Label-free quantification to measure protein abundance changes

  • SILAC or TMT labeling for more precise quantification

  • Focus on changes in:

    • Mitochondrial ribosomal proteins (particularly large subunit components)

    • Respiratory chain complex subunits (with special attention to Complex I)

    • Mitochondrial quality control machinery

• Targeted proteomics:

  • Selected/Multiple Reaction Monitoring (SRM/MRM) assays for precise quantification of key proteins

  • Parallel Reaction Monitoring (PRM) for improved specificity

Specialized Proteomics Techniques:

• Translatomics: Ribosome profiling of mitochondrial ribosomes to assess translation efficiency of specific mitochondrial mRNAs

• Complexome profiling:

  • Blue Native PAGE separation of intact complexes

  • Mass spectrometry analysis of gel slices

  • Provides insights into assembly intermediates and subcomplex accumulation

• Protein turnover analysis:

  • Pulse-chase SILAC to measure synthesis and degradation rates

  • Particularly informative for mitochondrial-encoded proteins

Data Analysis Framework:

• Pathway enrichment analysis to identify cellular processes affected beyond direct translation effects

• Correlation network analysis to identify co-regulated protein groups

• Integration with transcriptomics and metabolomics data for systems-level understanding

This proteomics strategy has been successfully applied to patient fibroblasts, revealing that MRPL50 deficiency leads to destabilization of the large mitochondrial ribosomal subunit while preserving the small subunit, with consequent effects on Complex I abundance .

What are the most common issues when using MRPL50 antibodies and how can they be resolved?

When working with MRPL50 antibodies, researchers may encounter several technical challenges. Here are solutions for the most common issues:

Issue 1: Weak or Absent Signal in Western Blot

Issue 2: Multiple Bands or Non-specific Binding

Issue 3: Inconsistent IHC Staining

Issue 4: High Background in Immunofluorescence

General Optimization Tips:

• Always include positive controls (HepG2, HeLa, HT-1080 cells)

• Titrate antibody for each application and sample type

• Follow recommended sample preparation procedures

• Consider alternative antibody clones if persistent issues occur

These troubleshooting strategies address the most common technical challenges encountered when working with MRPL50 antibodies in various research applications.

What are the key considerations for designing comprehensive MRPL50 research projects?

When designing comprehensive research projects focused on MRPL50, investigators should consider several critical factors to ensure robust experimental design and meaningful outcomes:

Biological Context and Significance:

• MRPL50 is a component of the mitochondrial ribosome large subunit with specific impacts on mitochondrial translation

• MRPL50 deficiency has been linked to a syndrome affecting high-energy demand tissues, highlighting its clinical relevance

• Research should consider both structural roles in ribosome assembly and potential specialized functions in translation regulation

Experimental Design Framework:

• Model selection: Choose appropriate models based on research questions:

  • Cell lines (HeLa, HepG2, HT-1080) for basic mechanistic studies

  • Patient-derived fibroblasts for disease-relevant phenotypes

  • Model organisms (Drosophila validated) for in vivo studies

  • Tissue-specific models for investigating organ-specific pathology

• Methodological approach: Implement multi-level analysis:

  • Molecular: Protein-protein interactions, ribosome assembly

  • Cellular: Mitochondrial translation, OXPHOS function

  • Physiological: Tissue-specific energetics and function

  • Systems: Integrative omics approaches

• Technical considerations:

  • Antibody selection and validation for specific applications

  • Appropriate controls for interpreting MRPL50 manipulation

  • Normalization strategies for mitochondrial content

  • Quantitative approaches for measuring mild phenotypes

Future Research Directions:

• Investigate tissue-specific requirements for MRPL50 function

• Explore potential specialized roles in translating specific mitochondrial mRNAs

• Identify therapeutic approaches for MRPL50-associated disorders

• Examine potential connections to broader mitochondrial pathologies

By integrating these considerations into research design, investigators can develop comprehensive projects that advance understanding of MRPL50's role in mitochondrial biology and disease pathogenesis, building upon the foundation established by recent discoveries linking MRPL50 deficiency to specific clinical syndromes .