MRPS25 Antibody

What is MRPS25 and what is its functional significance in mitochondrial biology?

MRPS25 (Mitochondrial Ribosomal Protein S25) is a critical structural component of the 28S small subunit of the mitochondrial ribosome. Unlike many mitochondrial ribosomal proteins, MRPS25 does not have a bacterial homolog, indicating it emerged during the evolutionary divergence of mitochondrial ribosomes from their bacterial origins .

The protein plays an essential role in mitochondrial translation by stabilizing the small ribosomal subunit. Research has demonstrated that MRPS25 deficiency leads to destabilization of the entire 28S subunit, subsequently impairing mitochondrial protein synthesis and reducing levels of respiratory chain components . This highlights its fundamental importance in cellular energy production and mitochondrial function.

Structurally, MRPS25 is closely positioned with other small subunit proteins like MRPS16 and MRPS22 in the assembled mitochondrial ribosome, suggesting potential functional interactions among these components .

How can researchers effectively distinguish between MRPS25 and other mitochondrial ribosomal proteins?

When investigating MRPS25, researchers must employ specific strategies to distinguish it from other mitochondrial ribosomal proteins:

• Antibody selection: Use antibodies targeting unique epitopes of MRPS25, such as those recognizing the C-terminal region (amino acids 104-173) or other specific sequences not conserved in related proteins.

• Molecular weight identification: MRPS25 has a characteristic molecular weight of approximately 18 kDa , which can help distinguish it from other mitochondrial ribosomal proteins during Western blot analysis.

• Immunogen sequence verification: When selecting antibodies, verify that the immunogen sequence is specific to MRPS25. For example, some validated antibodies use the sequence "KILGKNEERTLREEEEEKKQLSHPANFGPRKYCLRECICEV EGQVPCPSLVPLPKEMRGKYKAALKA DAQDĀ" , which corresponds to amino acids 104-173 of human MRPS25.

• Knockout/knockdown controls: Include MRPS25 knockout or knockdown samples as negative controls to confirm antibody specificity and distinguish from potential cross-reactivity with related proteins.

What are the validated applications for MRPS25 antibodies and how should experiments be designed?

Based on current research and commercial antibody validation data, MRPS25 antibodies have been successfully applied in the following experimental techniques:

When designing experiments:

• Include appropriate positive controls: HeLa, A-549, and BxPC-3 cell lines have been validated as positive controls for MRPS25 detection .

• Design rigorous validation protocols: Before proceeding with large-scale experiments, validate antibody specificity using complementary techniques (e.g., RNA interference, overexpression systems).

• Consider subcellular fractionation: Since MRPS25 is localized to mitochondria, enriching mitochondrial fractions may improve detection sensitivity in samples with low expression .

How can researchers accurately assess MRPS25 expression changes in disease models?

When investigating MRPS25 expression changes in disease models, researchers should implement the following methodological approaches:

• Quantitative Western blot analysis: Use densitometry with appropriate normalization controls. For mitochondrial proteins like MRPS25, normalization to total mitochondrial protein (e.g., VDAC or TOM20) rather than whole-cell housekeeping genes provides more accurate quantification.

• Multiple detection methods: Combine protein detection (Western blot) with mRNA quantification (qRT-PCR) to distinguish between transcriptional and post-transcriptional regulation.

• Control for mitochondrial content: Changes in MRPS25 levels might reflect altered mitochondrial content rather than specific regulation. Assess multiple mitochondrial markers from different compartments (matrix, membrane).

• Complementation studies: As demonstrated in research with MRPS25 mutations, complementation with wild-type MRPS25 provides compelling evidence for the specificity of observed defects . Rescue experiments should be included in disease models where MRPS25 dysfunction is suspected.

What strategies can resolve inconsistent MRPS25 detection in Western blot applications?

When encountering difficulties in MRPS25 detection by Western blot, consider these methodological solutions:

• Sample preparation optimization:

  • Ensure complete lysis of mitochondria using appropriate detergents (e.g., 1% Triton X-100 or RIPA buffer)

  • Add protease inhibitors immediately upon lysis

  • Avoid freeze-thaw cycles of prepared samples

• Transfer conditions adjustment:

  • For the 18 kDa MRPS25 protein, use PVDF membranes with 0.2 μm pore size rather than 0.45 μm

  • Consider semi-dry transfer systems for small proteins

  • Optimize transfer time to prevent protein loss

• Antibody incubation optimization:

  • Test multiple dilutions within the recommended range (1:500 - 1:2000)

  • Extend primary antibody incubation to overnight at 4°C

  • Use 5% BSA instead of milk for blocking and antibody dilution if background is high

• Verify sample integrity:

  • Run positive control samples (HeLa, A-549, BxPC-3) in parallel

  • Check for protein degradation using other mitochondrial marker antibodies

  • Consider using freshly prepared samples if archived samples yield inconsistent results

How should researchers interpret complex patterns of MRPS25 interaction with other mitochondrial ribosomal components?

Interpretation of MRPS25 interactions requires careful consideration of the following methodological approaches:

• Co-immunoprecipitation analysis: When performing co-IP experiments to study MRPS25 interactions:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Include RNase treatment controls to distinguish RNA-dependent from direct protein interactions

  • Consider crosslinking to capture transient interactions

• Data interpretation guidelines:

  • Research indicates that MRPS25 deficiency affects levels of other small subunit components (MRPS17, MRPS22, and MRPS29), while large subunit components (MRPL44 and MRPL45) remain relatively unaffected

  • Changes in 28S/39S subunit ratios may indicate compensatory or regulatory mechanisms

  • Consider indirect effects through altered ribosome assembly rather than direct interactions

• Validation through multiple techniques:

  • Complement co-IP results with proximity labeling techniques (BioID, APEX)

  • Use sucrose gradient fractionation to analyze intact ribosomal complexes

  • Consider cryo-EM studies for structural verification of interaction sites

How can MRPS25 antibodies be utilized to study mitochondrial disease mechanisms?

Research into MRPS25-related pathologies can be enhanced through these methodological approaches:

• Tissue-specific expression analysis:

  • Analyze MRPS25 expression across different tissues relevant to mitochondrial diseases (brain, muscle, heart)

  • Compare expression patterns in affected versus unaffected tissues in disease models

  • Correlate MRPS25 levels with disease severity markers

• Functional impact assessment:

  • Use MRPS25 antibodies to monitor ribosome assembly states in patient-derived samples

  • Implement pulse-labeling experiments to quantify mitochondrial translation efficiency

  • Assess respiratory chain complex assembly through BN-PAGE combined with Western blot

• Therapeutic monitoring:

  • Track MRPS25 levels and mitochondrial ribosome assembly during experimental treatments

  • Monitor mitochondrial translation recovery in complementation studies

  • Develop quantitative assays for high-throughput screening applications

Research has demonstrated that mutations in MRPS25 (e.g., p.P72L) can lead to mitochondrial disease with clinical features including dyskinetic cerebral palsy and partial agenesis of the corpus callosum . Patient fibroblasts showed approximately 10-fold reduction in MRPS25 protein levels, destabilization of the small ribosomal subunit, impaired mitochondrial translation, and reduced levels of respiratory chain components .

What are the methodological considerations when investigating the role of MRPS25 in mitochondrial ribosome assembly?

When studying MRPS25's role in mitochondrial ribosome assembly, researchers should consider these approaches:

• Sucrose gradient analysis protocol:

  • Prepare mitochondrial lysates with gentle detergents to preserve ribosomal complexes

  • Load equal amounts of mitochondrial protein on 10-30% sucrose gradients

  • Centrifuge at 100,000 × g for 2-3 hours

  • Collect fractions and analyze by Western blot for:

    • MRPS25 and other small subunit proteins (28S fraction)

    • Large subunit proteins (39S fraction)

    • Assembled mitoribosome (55S fraction)

• Quantitative assessment of assembly:

  • Measure 12S and 16S rRNA levels as indicators of small and large subunit integrity

  • Track distribution shifts of ribosomal proteins across gradient fractions

  • Compare assembly patterns between normal and MRPS25-deficient samples

• Structure-function analysis:

  • Use site-directed mutagenesis to introduce specific mutations (e.g., P72L) identified in patients

  • Assess the impact on protein stability and ribosome assembly

  • Perform complementation studies with wild-type MRPS25 to confirm specificity of observed defects

Research has shown that MRPS25 deficiency affects the assembly of the entire 28S subunit, with consequent effects on mitochondrial translation . Experiments with the P72L mutation demonstrated decreased levels of multiple small subunit components and impaired translation of mitochondrially-encoded proteins .

How might MRPS25 antibodies be applied to study the relationship between mitochondrial translation and neurodevelopmental disorders?

Recent research linking MRPS25 mutations to cerebral palsy and corpus callosum abnormalities suggests several methodological approaches:

• Neuronal model systems:

  • Utilize MRPS25 antibodies in iPSC-derived neuronal cultures from patients or gene-edited models

  • Develop protocols for simultaneous detection of MRPS25 and neuronal differentiation markers

  • Implement spatial analysis techniques to assess MRPS25 distribution in neuronal compartments

• Developmental timing studies:

  • Design experiments to track MRPS25 expression during critical periods of neurodevelopment

  • Correlate MRPS25 levels with mitochondrial translation activity in developing neurons

  • Analyze the impact of MRPS25 deficiency on energy-demanding neurodevelopmental processes

• Tissue-specific analysis in model organisms:

  • Develop protocols for region-specific analysis of MRPS25 expression in brain tissues

  • Implement multiplexed detection methods to correlate MRPS25 with markers of corpus callosum development

  • Use longitudinal sampling approaches to track changes throughout development

Research has revealed that defects in small subunit components of mitochondrial ribosomes, including MRPS25, should be considered when defects of the corpus callosum are present in patients . This finding underscores the importance of mitochondrial translation in neurodevelopment and suggests new diagnostic approaches for neurodevelopmental disorders.

What are the methodological considerations for investigating potential tissue-specific functions of MRPS25?

When exploring potential tissue-specific functions of MRPS25, researchers should implement:

• Comparative expression analysis protocol:

  • Prepare tissue samples with standardized extraction methods to maintain protein integrity

  • Include multiple antibodies targeting different MRPS25 epitopes to confirm specificity

  • Normalize expression to tissue-specific mitochondrial content markers

  • Compare relative abundance across tissues (brain, heart, skeletal muscle, liver, kidney)

• Isoform detection strategies:

  • Design primers and antibodies capable of distinguishing potential isoforms

  • Implement RT-PCR to detect tissue-specific transcript variants before protein analysis

  • Consider IP-mass spectrometry approaches to identify potential post-translational modifications

• Functional analysis considerations:

  • Develop tissue-specific knockout or knockdown models

  • Compare mitochondrial translation efficiency across tissues with equivalent MRPS25 reduction

  • Assess compensation mechanisms that may differ between tissues

Evidence suggests tissue-specific vulnerability to MRPS25 dysfunction, with brain involvement (structural or functional) being common among disorders arising from mutations in mitochondrial ribosomal proteins . While cardiomyopathy has been reported in some MRP gene mutations, it was not evident in the case of MRPS25 P72L mutation .

What are the critical validation steps for confirming MRPS25 antibody specificity in research applications?

To ensure reliable results with MRPS25 antibodies, implement these validation protocols:

• Comprehensive specificity testing:

  • Perform Western blot analysis on positive control cell lines (HeLa, A-549, BxPC-3)

  • Include genetic controls (MRPS25 knockout/knockdown) where available

  • Test for cross-reactivity with recombinant MRPS25 protein

• Epitope verification:

  • Confirm recognition of the expected epitope through peptide competition assays

  • For antibodies targeting C-terminal regions , verify detection of this region in your experimental system

  • Consider multiple antibodies recognizing different epitopes for confirmation

• Application-specific validation:

  • For Western blot: Confirm single band at expected molecular weight (18 kDa)

  • For IP: Verify enrichment through Western blot of input, unbound, and eluted fractions

  • For ELISA: Establish standard curves with recombinant protein

• Correlation with orthogonal methods:

  • Compare protein detection results with mRNA quantification data

  • Validate subcellular localization through fractionation studies

  • Confirm functional outcomes through mitochondrial translation assays

How can researchers design definitive experiments to establish causality between MRPS25 dysfunction and observed phenotypes?

To establish causality between MRPS25 dysfunction and experimental phenotypes:

• Complementation study design:

  • Introduce wild-type MRPS25 into deficient cells through appropriate expression systems

  • Include proper controls (empty vector, unrelated protein expression)

  • Quantify rescue of:

    • MRPS25 protein levels

    • Small ribosomal subunit assembly

    • Mitochondrial translation

    • OXPHOS protein levels

    • Phenotypic outcomes

• Dose-response relationships:

  • Develop graded knockdown systems to establish correlation between MRPS25 levels and severity of phenotypes

  • Implement inducible expression systems to determine threshold effects

  • Quantify multiple downstream markers to establish hierarchical relationships

• Mutation-specific studies:

  • Compare effects of different mutations (e.g., P72L versus other pathogenic variants)

  • Analyze structure-function relationships based on location within the protein

  • Correlate biochemical defects with clinical severity

Research has demonstrated that lentiviral-mediated expression of wild-type MRPS25 in cells with the MRPS25-P72L variant restored MRPS levels, 28S subunit assembly, and partially restored OXPHOS protein levels, establishing the disease-causing nature of the mutation . Interestingly, expression of MRPS25 in control cells resulted in marked cell death, suggesting a possible toxic effect in the context of normal 28S subunit assembly .