MRPL49 Antibody

What is MRPL49 and why is it important for mitochondrial function?

MRPL49 (Mitochondrial Ribosomal Protein L49) is an essential component of the 39S large subunit of the mitochondrial ribosome (mitoribosome). The mitoribosome is a 55S ribonucleoprotein complex composed of large and small subunits that coordinates the synthesis of the 13 proteins encoded by the mitochondrial genome . These proteins are vital components of the oxidative phosphorylation (OXPHOS) enzyme complexes.

Unlike bacterial, chloroplast, and cytosolic ribosomes, MRPL49 has no apparent homolog in these systems, suggesting a unique role in mitochondrial translation . Current research indicates that MRPL49:

• May compensate for lost rRNA and stabilize bypass segments within the mt-LSU

• Interacts intricately with MRPL4, MRPL15, MRPL57, and MRPL64, alongside the 16S rRNA

• Is critical for maintaining the proper three-dimensional architecture of the mitoribosome

• Has a homolog in yeast (Img2) that is required for mitochondrial genome integrity

The importance of MRPL49 is highlighted by the fact that biallelic variants in the MRPL49 gene cause combined oxidative phosphorylation deficiency (COXPD), a rare multisystem disorder .

When using MRPL49 antibodies in Western blot applications, you should expect to observe a band at approximately 19 kDa, which corresponds to the calculated molecular weight of the protein . This has been consistently observed across multiple antibody sources.

Regarding cellular localization:

• MRPL49 is primarily localized in the mitochondrion

• In immunofluorescence studies, MRPL49 shows a punctate cytoplasmic pattern consistent with mitochondrial localization

• In tissue sections from wild-type adult mice, MRPL49 has been observed in the mitochondria of outer hair cells, inner hair cells, and supporting cells of the inner ear

For optimal detection in immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may also be used as an alternative .

How can I use MRPL49 antibodies to study mitochondrial translation defects?

MRPL49 antibodies can be instrumental in investigating mitochondrial translation defects through several sophisticated approaches:

Complexome Profiling Method:
Complexome profiling is a quantitative mass spectrometry approach that can characterize deficiencies in respiratory complex genes . The methodology involves:

• Isolating enriched mitochondrial fractions from patient-derived or experimental cell lines

• Performing Blue Native electrophoresis (BNE)

• Systematic dissection of the polyacrylamide gel

• Tryptic digestion and tandem MS analysis

• Visualization of protein abundance in each discrete section as heatmaps and line charts

This approach can reveal alterations in the content of mitoribosomes, as evidenced by changes in levels of individual mt-LSU and mt-SSU components. In fibroblasts from patients with MRPL49 variants, complexome profiling revealed:

• Decreases of ~15-40% in mt-SSU levels

• More pronounced reductions of ~60-70% in mt-LSU levels

• Significant reductions in complex I and IV levels

Mitochondrial Protein Translation Assays:
To examine the impact of MRPL49 dysfunction on mitochondrial protein synthesis:

• Measure the activity and levels of OXPHOS complexes using MRPL49 antibodies for immunoblotting

• Compare complex I and IV subunit levels between control and experimental samples

• Correlate changes in OXPHOS subunit levels with clinical phenotypes

16S:12S rRNA Ratio Analysis:
Previous studies investigating disease-associated variants in genes encoding mt-LSU proteins have consistently demonstrated a relative reduction in 16S rRNA levels . Using RT-qPCR in conjunction with MRPL49 antibody validation:

• Extract RNA from control and experimental cells

• Perform RT-qPCR to quantify 16S and 12S rRNA levels

• Calculate the 16S:12S rRNA ratio

• Correlate changes in this ratio with MRPL49 protein levels detected by antibodies

This approach can provide insights into how MRPL49 variants affect mitoribosome stability and function.

What controls and validation steps should I use to ensure MRPL49 antibody specificity?

Ensuring antibody specificity is critical for obtaining reliable research results. For MRPL49 antibodies, consider implementing the following validation strategy:

Essential Controls:

• Positive Controls:

  • Use cell lines with confirmed MRPL49 expression (BxPC-3, A549, or HeLa cells have been validated)

  • Include recombinant MRPL49 protein as a standard

• Negative Controls:

  • MRPL49 knockdown or knockout cells (using siRNA or CRISPR-Cas9)

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

  • Isotype control to evaluate background

• Peptide Competition Assay:

  • Pre-incubate the antibody with blocking peptide corresponding to the immunogen

  • Run parallel experiments with blocked and unblocked antibody

  • The specific signal should be significantly reduced or eliminated with the blocking peptide

Advanced Validation Methods:

• Orthogonal Validation:

  • Confirm results using at least two antibodies targeting different epitopes of MRPL49

  • Compare protein expression with mRNA levels using qPCR

• Cross-Reactivity Assessment:

  • Test antibody against related mitochondrial ribosomal proteins, particularly those with similar molecular weights

  • Evaluate performance across multiple species if cross-reactivity is claimed

• Application-Specific Validation:

  • For IHC: Test multiple fixation methods and antigen retrieval protocols

  • For IP: Confirm pulled-down protein by mass spectrometry

  • For WB: Verify band size and use appropriate loading controls

Technical Considerations:
When using MRPL49 antibodies for Western blot, the sensitivity of different assays can vary. This has been demonstrated in fibroblasts from patients with MRPL49 variants, where complex I subunit deficiencies were detected in enzymatic assays but not always visible by Western blot .

How do MRPL49 expression patterns correlate with mitochondrial disease phenotypes?

MRPL49 dysregulation has been implicated in a spectrum of mitochondrial diseases, particularly combined oxidative phosphorylation deficiency (COXPD). Recent research has revealed fascinating correlations between MRPL49 expression patterns and disease phenotypes:

Phenotypic Spectrum Associated with MRPL49 Variants:

Genotype-Phenotype Correlations:
Interestingly, even within families harboring identical MRPL49 variants (e.g., homozygous His92Pro), there can be striking inter-familial phenotypic differences. For example:

• Some affected females present with classical features of Perrault syndrome

• Others with the same variant show no evidence of ovarian insufficiency or hearing loss

This suggests the presence of undefined genetic modifiers influencing the phenotypic expression of MRPL49 deficiency.

Correlation with Mitochondrial Function:
The severity of clinical presentation correlates with the degree of mitochondrial dysfunction as measured by:

• Reduction in mt-LSU and mt-SSU levels

• Decreased levels of OXPHOS enzyme complexes I and IV

• Alterations in the 16S:12S rRNA ratio

For example, in fibroblasts from a severely affected individual (F5:II-1), complexome profiling revealed more pronounced reductions in mitoribosomal components (~40% in mt-SSU and 70% in mt-LSU) compared to a less severely affected individual (F1:II-1, ~15% in mt-SSU and 60% in mt-LSU) .

Research Implications:
When using MRPL49 antibodies to study these correlations, researchers should:

• Quantify MRPL49 protein levels in patient-derived cells or tissues

• Correlate protein levels with disease severity and specific phenotypes

• Consider the impact of genetic background on phenotypic expression

What are the optimal protocols for using MRPL49 antibodies in complexome profiling studies?

Complexome profiling is a powerful approach for analyzing mitochondrial protein complexes and has been successfully applied to study MRPL49-associated pathologies. Here is a detailed protocol optimized for MRPL49 antibody use in complexome profiling:

Sample Preparation:

• Enrich mitochondrial fractions from cultured cells (fibroblasts or other cell types) :

  • Homogenize cells in isolation buffer (250 mM sucrose, 10 mM Tris-HCl pH 7.5, 1 mM EDTA)

  • Centrifuge at 1,000 × g for 10 minutes to remove nuclei and unbroken cells

  • Centrifuge supernatant at 10,000 × g for 10 minutes to pellet mitochondria

  • Wash mitochondrial pellet twice with isolation buffer

• Solubilize mitochondrial proteins:

  • Resuspend mitochondrial pellet in 50 mM NaCl, 50 mM imidazole, 2 mM aminohexanoic acid, 1 mM EDTA, pH 7.0

  • Add digitonin or n-dodecyl-β-D-maltoside at a detergent-to-protein ratio of 4:1

  • Incubate on ice for 10 minutes

  • Centrifuge at 20,000 × g for 20 minutes to remove insoluble material

Blue Native Electrophoresis (BNE):

• Load solubilized proteins onto 3-12% or 4-16% gradient native PAGE gels

• Run at 100 V for 30 minutes, then increase to 300 V, maintaining current below 15 mA

• Use Coomassie Blue G-250 as the charge carrier in cathode buffer

Sample Processing for Mass Spectrometry:

• Cut gel lanes into approximately 60 equal pieces (1 mm each)

• Process each gel piece separately:

  • Destain with 50% acetonitrile in 50 mM ammonium bicarbonate

  • Reduce with 10 mM DTT for 30 minutes at 56°C

  • Alkylate with 55 mM iodoacetamide for 30 minutes in the dark

  • Digest with trypsin overnight at 37°C

  • Extract peptides with 50% acetonitrile/0.1% formic acid

  • Dry extracts and reconstitute in 0.1% formic acid

Mass Spectrometry Analysis:

• Analyze samples using nano-LC-MS/MS

• Use a standard 90-minute gradient for peptide separation

• Process data using MaxQuant or similar software for protein identification

• Normalize protein intensities to allow comparison between samples

MRPL49 Antibody Validation During Complexome Profiling:

• Run a parallel Western blot of the same BN-PAGE gel

• Transfer proteins to PVDF membrane

• Probe with MRPL49 antibody (recommended dilution: 1:500)

• Compare MRPL49 migration pattern with MS-based complexome profile

• Use known mitoribosomal markers (e.g., MRPL4, MRPL15) as references

Data Analysis and Visualization:

• Generate heatmaps and line charts to visualize protein abundance across gel slices

• Specifically analyze:

  • MRPL49 distribution pattern

  • Co-migration with other mt-LSU components

  • Relative abundance compared to control samples

  • Correlation with OXPHOS complex profiles

Special Considerations:

• Deposit MS data to repositories like ProteomeXchange Consortium via PRIDE

• Include dataset identifiers in publications for reproducibility

• For MRPL49 variant studies, analyze how variants affect migration patterns of interacting partners (MRPL4, MRPL15, MRPL57, MRPL64)

How can I use MRPL49 antibodies to investigate mitochondrial disease mechanisms?

MRPL49 antibodies serve as valuable tools for elucidating the molecular mechanisms underlying mitochondrial diseases, particularly those involving mitoribosomal dysfunction. Here are methodological approaches for using these antibodies in mitochondrial disease research:

1. Characterizing Pathogenic Variants Impact:

To investigate how MRPL49 variants affect protein function and mitochondrial translation:

• Structure-Function Analysis:

  • Use immunoprecipitation with MRPL49 antibodies followed by mass spectrometry to identify altered protein interactions

  • Focus on interactions with key partners (MRPL4, MRPL15, MRPL57, MRPL64) and 16S rRNA

  • Compare wild-type vs. variant MRPL49 binding patterns

• Mitoribosome Assembly Assessment:

  • Perform sucrose gradient centrifugation to separate ribosomal subunits

  • Use MRPL49 antibodies in Western blot to track protein distribution across fractions

  • Compare assembly profiles between wild-type and disease models

2. Translational Defect Quantification:

• Pulse-Chase Labeling:

  • Perform [35S]-methionine labeling of newly synthesized mitochondrial proteins

  • Immunoprecipitate MRPL49 to assess its association with active translation

  • Quantify translation rates in the presence of wild-type vs. variant MRPL49

• Polysome Profiling:

  • Isolate mitochondria and extract polysomes

  • Use MRPL49 antibodies to track mitoribosome distribution

  • Compare polysome profiles between control and disease models

3. Tissue-Specific Pathology Investigation:

Recent findings show that MRPL49 deficiency presents with tissue-specific manifestations . To investigate this:

• Immunohistochemistry Protocol Optimization:

  • For brain tissue (to study leukodystrophy pathology):

    • Use antigen retrieval with TE buffer pH 9.0

    • Recommended antibody dilution: 1:50-1:200

    • Counterstain with DAPI to visualize nuclei

  • For inner ear tissue (to study hearing loss mechanism):

    • Decalcify temporal bones prior to processing

    • Use MRPL49 antibody at 1:50 dilution

    • Co-stain with markers for hair cells (Myo7a) and mitochondria (TOMM20)

  • For retinal tissue (to study retinal dystrophy):

    • Cryosection eyes at 10-12 μm thickness

    • Use MRPL49 antibody at 1:50 dilution

    • Co-stain with photoreceptor markers

4. Therapeutic Strategy Evaluation:

MRPL49 antibodies can be used to assess the efficacy of experimental treatments:

• Gene Therapy Monitoring:

  • Quantify MRPL49 protein levels before and after gene supplementation

  • Assess restoration of mitoribosome assembly and function

  • Correlate with phenotypic rescue

• Small Molecule Screening:

  • Use MRPL49 antibodies in high-throughput immunofluorescence assays

  • Screen compounds that stabilize mutant MRPL49 or enhance residual mitoribosome function

  • Validate hits with secondary assays measuring OXPHOS complex formation

5. Disease Progression Biomarkers:

MRPL49 antibodies may help identify biomarkers for disease progression:

• Longitudinal Analysis:

  • Measure MRPL49 levels in accessible tissues (e.g., fibroblasts, blood cells)

  • Correlate with disease severity and progression

  • Establish threshold values for clinical significance

Methodological Guidance for Brain MRI Correlation:
MRPL49-associated pathologies show distinctive brain MRI findings, particularly symmetrical involvement of the globi pallidi and deep white matter changes . Researchers can correlate these imaging findings with molecular changes by:

• Performing immunohistochemistry on post-mortem brain tissue from affected regions

• Using MRPL49 antibodies to assess protein localization and abundance

• Correlating findings with specific MRI patterns (e.g., T2 high signal, diffusion restriction, cystic change)

• Comparing results across disease stages to understand progressive pathology

What are the technical considerations for troubleshooting inconsistent results with MRPL49 antibodies?

When facing inconsistent results with MRPL49 antibodies, a systematic troubleshooting approach is essential. Below are methodological solutions to common technical challenges:

1. Variability in Western Blot Detection:

Technical Note: The sensitivity of different assays can vary significantly. In fibroblasts from patients with MRPL49 variants, complex I subunit deficiencies were detected in enzymatic assays but not always visible by Western blot .

2. Immunohistochemistry Optimization:

MRPL49 detection in tissues requires careful optimization:

• Fixation Effects:

  • Overfixation in formalin can mask epitopes

  • Test multiple fixation times (6, 12, 24 hours)

  • Compare frozen vs. FFPE sections for epitope preservation

• Antigen Retrieval Comparison:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative option)

  • EDTA buffer pH 8.0

  • Test multiple heat sources (microwave, pressure cooker, water bath)

• Antibody Incubation Optimization:

  • Test different temperatures (4°C, room temperature, 37°C)

  • Vary incubation times (overnight, 1 hour, 2 hours)

  • Dilution series from 1:50 to 1:500

3. Cross-Reactivity Assessment:

MRPL49 has pseudogenes located on chromosomes 5q and 8p , which may affect antibody specificity:

• Perform BLAST analysis of the immunogen sequence

• Test antibody in cells with MRPL49 knockout

• Compare results with antibodies targeting different epitopes

4. Sample-Specific Considerations:

Different sample types require specific handling for optimal MRPL49 detection:

• Cell Lines:

  • Validated positive controls include BxPC-3, A549, and HeLa cells

  • Synchronize cells to control for cell cycle-dependent expression

  • Enrich mitochondrial fraction for enhanced detection

• Tissue Samples:

  • Fresh tissues should be fixed within 30 minutes of collection

  • Consider regional variation in mitochondrial content

  • Use FFPE samples less than 5 years old for consistent results

• Patient-Derived Samples:

  • Account for genetic background variations

  • Consider disease state and medication effects

  • Compare with age-matched controls

5. Complexome Profiling Challenges:

For researchers using MRPL49 antibodies in complexome profiling studies:

• Ensure complete solubilization of mitochondrial membranes

• Optimize detergent type and concentration (digitonin vs. n-dodecyl-β-D-maltoside)

• Use a mitochondrial marker protein as an internal standard

• Consider native vs. denaturing conditions based on research question

6. Advanced Analytical Approach:

When troubleshooting particularly difficult cases:

• Quantify mitochondrial DNA content to normalize for mitochondrial mass

• Measure 16S:12S rRNA ratio to assess mt-LSU stability

• Perform RT-qPCR to compare mRNA and protein levels

• Consider post-translational modifications or alternative splicing

How can MRPL49 antibodies be used in studying tissue-specific mitochondrial translation regulation?

Recent research has revealed intriguing tissue-specific manifestations of MRPL49 deficiency, suggesting differential regulation of mitochondrial translation across tissues. MRPL49 antibodies can be instrumental in exploring these tissue-specific mechanisms:

Methodological Approach for Tissue Comparison:

• Multi-tissue Expression Analysis:

  • Perform Western blot analysis of MRPL49 across tissue panels

  • Quantify relative expression levels normalized to tissue-specific housekeeping proteins

  • Compare with other mitoribosomal proteins to identify tissue-specific patterns

  Recent immunofluorescence studies in wild-type adult mice showed MRPL49 expression in the mitochondria of outer hair cells, inner hair cells, and supporting cells of the inner ear . This tissue-specific expression pattern provides insights into the hearing loss phenotype observed in some patients with MRPL49 variants.

• Cell Type-Specific Translation Assessment:

  • Use immunofluorescence co-localization with cell type-specific markers

  • Combine with mitochondrial protein synthesis assays

  • Quantify translation efficiency in different cell populations

• Developmental Regulation Analysis:

  • Track MRPL49 expression during embryonic and post-natal development

  • Correlate with mitochondrial maturation milestones

  • Identify critical periods for tissue-specific mitochondrial biogenesis

Experimental Design for Tissue-Specific Studies:

For investigating the role of MRPL49 in tissue-specific pathologies:

• Neurological Manifestations:

  • Compare MRPL49 levels in different brain regions (cerebellum, basal ganglia, white matter)

  • Correlate with region-specific vulnerability in leukodystrophy

  • Use primary neuronal cultures to assess cell autonomous effects

• Reproductive System:

  • Analyze MRPL49 expression in ovarian tissue at different developmental stages

  • Correlate with markers of ovarian reserve and folliculogenesis

  • Investigate the mechanistic link to primary ovarian insufficiency

• Sensory Systems:

  • Compare MRPL49 distribution in cochlear vs. retinal tissues

  • Assess mitochondrial content and activity in these specialized cells

  • Explore relationships with tissue-specific energy demands

Advanced Single-Cell Applications:

To dissect cell type-specific roles of MRPL49:

• Combine MRPL49 immunostaining with laser capture microdissection

• Perform single-cell proteomics on isolated populations

• Correlate MRPL49 levels with mitochondrial function at single-cell resolution

What is the role of MRPL49 in modulating mitochondrial stress responses?

The mitochondrial ribosome plays a crucial role in mitochondrial stress responses, and MRPL49 may serve as a key regulatory component. MRPL49 antibodies can help elucidate these mechanisms:

Methodological Framework for Stress Response Studies:

• Stress Induction Protocols:

  • Oxidative stress: Treatment with hydrogen peroxide or paraquat

  • Protein misfolding stress: Treatment with tunicamycin or thapsigargin

  • Mitochondrial translation stress: Treatment with doxycycline or chloramphenicol

  • Energy depletion: Glucose deprivation or oligomycin treatment

• MRPL49 Response Assessment:

  • Monitor MRPL49 protein levels by Western blot at different time points post-stress

  • Track subcellular localization changes by immunofluorescence

  • Assess post-translational modifications using specialized antibodies

• Integration with Stress Response Pathways:

  • Co-immunoprecipitation of MRPL49 with stress response mediators

  • Analysis of MRPL49 association with quality control machinery

  • Investigation of potential regulatory interactions

Experimental Design for Patient-Derived Cells:

For cells harboring MRPL49 variants, stress response studies can provide insights into disease pathomechanisms:

• Expose control and patient fibroblasts to graded stressors

• Monitor activation of mitochondrial stress responses:

  • Mitochondrial unfolded protein response (UPRmt)

  • Integrated stress response (ISR)

  • Mitophagy and quality control pathways

• Correlate stress susceptibility with clinical severity

Based on complexome profiling data, fibroblasts from severely affected individuals show more pronounced reductions in mitoribosomal components compared to less severely affected individuals . This suggests differential stress response capabilities that could be further explored using MRPL49 antibodies.

How can MRPL49 antibodies contribute to understanding mitochondrial disease heterogeneity?

The variable clinical presentations associated with MRPL49 variants highlight the complex interplay between genotype and phenotype in mitochondrial diseases. MRPL49 antibodies can play a crucial role in unraveling this heterogeneity:

Methodological Strategy for Heterogeneity Analysis:

• Genotype-Phenotype Correlation:

  • Quantify MRPL49 protein levels in patient-derived cells

  • Correlate with specific variant types and positions

  • Relate to clinical severity and organ involvement

• Modifier Detection:

  • Develop co-immunoprecipitation protocols to capture MRPL49 interactors

  • Identify variant-specific interaction patterns

  • Screen for genetic or environmental modifiers that alter MRPL49 function

• Threshold Effect Investigation:

  • Establish dose-response relationships between MRPL49 levels and mitochondrial function

  • Determine tissue-specific thresholds for functional impairment

  • Create models for predicting phenotypic severity

Experimental Approach for Variability Studies:

The striking inter-familial differences observed even with identical MRPL49 variants (e.g., homozygous His92Pro) suggest the presence of undefined genetic modifiers . To investigate this:

• Create a panel of cell lines with controlled genetic backgrounds

• Introduce identical MRPL49 variants

• Compare protein levels, stability, and function using MRPL49 antibodies

• Correlate with mitochondrial translation efficiency and OXPHOS assembly

This approach can help identify factors contributing to the phenotypic variability observed in MRPL49-associated disorders, ranging from classical Perrault syndrome to severe childhood-onset leukodystrophy .

What are the future directions for MRPL49 antibody applications in mitochondrial research?

As our understanding of mitochondrial biology continues to evolve, MRPL49 antibodies will play an increasingly important role in advancing several frontier areas of research:

• Single-cell mitochondrial heterogeneity studies:

  • Combining MRPL49 antibodies with single-cell technologies to understand cell-to-cell variation in mitochondrial translation

  • Correlating mitoribosome composition with functional states in individual cells

• Therapeutic monitoring in mitochondrial disease trials:

  • Using MRPL49 antibodies as pharmacodynamic biomarkers for treatments targeting mitochondrial translation

  • Developing standardized assays for clinical trial endpoints

• Spatiotemporal regulation of mitochondrial translation:

  • Applying super-resolution microscopy with MRPL49 antibodies to visualize translation sites within mitochondria

  • Tracking dynamic changes in mitoribosome distribution during cellular stress

• Integration with multi-omics approaches:

  • Combining MRPL49 immunoprecipitation with RNA-seq to identify associated transcripts

  • Correlating proteomics and translatomics data to build comprehensive models of mitochondrial translation regulation