MRPS34 Antibody

What is MRPS34 and why is it significant in mitochondrial research?

MRPS34 (Mitochondrial Ribosomal Protein S34) is a key component of the small (28S) subunit of the mitochondrial ribosome. This protein plays a crucial role in the translation of mitochondrially-encoded polypeptides essential for oxidative phosphorylation and ATP production. Research has shown that MRPS34 is required for the stability of the 12S rRNA, the small ribosomal subunit, and actively translating ribosomes .

The significance of MRPS34 in mitochondrial research stems from its essential role in mitochondrial protein synthesis. Disruptions in MRPS34 function have been linked to mitochondrial dysfunction and metabolic disorders, highlighting its importance in cellular energy metabolism . Studies in mutant mice have demonstrated that compromised MRPS34 function leads to reduced levels of mitochondrial proteins and complexes, decreased oxygen consumption, and reduced respiratory complex activity, resulting in tissue-specific pathology .

What is the molecular weight of MRPS34 and how can I confirm proper detection?

According to experimental data, MRPS34 has both a calculated and observed molecular weight of 26 kDa . When performing Western blot analysis, researchers should expect to visualize a band at approximately this weight.

To confirm proper detection and antibody specificity:

• Run appropriate positive controls (A549, HeLa, or Jurkat cells are recommended)

• Include negative controls such as MRPS34 knockdown samples when available

• Verify that the band appears at the expected 26 kDa position

• Cross-validate results using different MRPS34 antibodies when possible

• Consider testing in multiple species if conducting comparative studies (verified reactivity includes human, mouse, and rat)

Which tissues express MRPS34 and at what levels?

MRPS34 is expressed in a wide range of tissues, though at varying levels. According to research findings, MRPS34 protein expression has been detected in:

When investigating tissue-specific expression patterns, it's important to note that the effects of MRPS34 mutations or dysfunction can vary significantly between tissues. For instance, research has shown that liver tissue often exhibits more pronounced molecular changes compared to cardiac tissue in MRPS34 mutant models .

What are the optimal conditions for Western blot applications using MRPS34 antibodies?

For optimal Western blot results with MRPS34 antibodies, researchers should follow these experimental conditions:

Optimized Western blot protocol:

• Load 20-40 μg of total protein per lane

• Separate proteins using 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membrane

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

• Incubate with primary antibody overnight at 4°C using the recommended dilution

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence detection

• Verify signal at expected 26 kDa molecular weight

For quantitative analysis, include appropriate loading controls and perform at least three biological replicates to ensure statistical robustness .

How can MRPS34 antibodies be optimized for immunohistochemistry applications?

For successful immunohistochemistry (IHC) with MRPS34 antibodies, consider these methodological recommendations:

Recommended IHC protocol:

• Tissue preparation: Use formalin-fixed, paraffin-embedded sections of 4-6 μm thickness

• Antigen retrieval: Use TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

• Antibody dilution: For 15166-1-AP, use 1:250-1:1000

• Positive control tissue: Human breast cancer tissue is recommended

• Detection system: Use a polymer-based detection system for enhanced sensitivity

• Counterstain: Hematoxylin for nuclear visualization

• Interpretation: Look for cytoplasmic staining consistent with mitochondrial localization

When interpreting IHC results, be aware that staining intensity may vary between different tissues due to varying levels of MRPS34 expression. Always include positive and negative controls to validate staining specificity.

What troubleshooting strategies can be employed when MRPS34 antibodies yield inconsistent results?

When facing inconsistent results with MRPS34 antibodies, consider these methodological troubleshooting approaches:

For weak or absent signal:

• Increase antibody concentration (within recommended ranges)

• Extend primary antibody incubation time (up to overnight at 4°C)

• Optimize antigen retrieval conditions (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

• Use signal amplification systems (e.g., tyramide signal amplification)

• Confirm sample integrity and protein expression using RT-qPCR

For high background or non-specific binding:

• Increase blocking time or concentration (5-10% blocking agent)

• Extend washing steps (5-6 washes of 5 minutes each)

• Reduce antibody concentration

• Filter antibody solution before use

• Include additional blocking agents (e.g., 0.1-0.3% Triton X-100)

For tissue-specific variations:

• Adjust protocols based on tissue type (e.g., longer antigen retrieval for fibrous tissues)

• Validate with multiple detection methods (WB, IF, IHC)

• Consider tissue-specific expression levels when interpreting results

For batch-to-batch variability:

• Validate each new lot against previous lots using known positive samples

• Maintain consistent experimental conditions across experiments

• Consider pooling antibody lots for long-term studies

How do MRPS34 mutations affect mitochondrial function in different tissues?

Research on MRPS34 mutant mice has revealed tissue-specific effects of MRPS34 dysfunction on mitochondrial function:

Molecular consequences of MRPS34 mutation:

• Ribosomal stability effects:

  • Decreased stability of the 12S rRNA in both heart and liver tissues

  • Compromised small ribosomal subunit stability

  • Reduced formation of actively translating ribosomes

• Tissue-specific effects on mitochondrial RNA:

  • Young mutant mice: mt-Nd5 mRNA levels decreased in liver but not heart

  • Aged mutant mice: mt-Co1, mt-Nd1, and mt-Nd5 mRNAs decreased in liver, while only mt-Nd5 decreased in heart

• Protein synthesis impairment:

  • Decreased synthesis of all 13 mitochondrially-encoded polypeptides

  • Initial rate of translation faster in control liver compared to heart mitochondria

  • More severe translation defects in liver compared to heart tissue

• Respiratory complex deficiencies:

  • Heart: Reduction in Complexes I and IV in both young and aged mice

  • Liver: Reduction in Complexes I, III, IV and V

  • More significant complex reduction in liver compared to heart tissue

These tissue-specific differences highlight the importance of considering tissue context when studying mitochondrial dysfunction and potential therapeutic interventions.

How can MRPS34 antibodies be used to investigate age-related mitochondrial dysfunction?

MRPS34 antibodies provide valuable tools for investigating age-related mitochondrial dysfunction through several methodological approaches:

Experimental designs for age-related studies:

• Longitudinal expression analysis:

  • Use Western blotting with MRPS34 antibodies to compare protein levels across different age groups

  • Correlate MRPS34 expression with markers of mitochondrial function (e.g., respiratory complex abundances, ATP production)

• Tissue-specific progression analysis:

  • Research has shown that molecular defects in MRPS34 mutant mice become more pronounced with age

  • In aged mutant mice, there is a more significant decrease in mitochondrially-encoded proteins (COXI, COXII) compared to young mutant mice

  • These effects appear to be cumulative over time and exhibit tissue-specific patterns

• Correlative analysis with functional parameters:

  • Combine MRPS34 protein level data with measurements of:

    • Oxygen consumption rates

    • ATP production

    • Mitochondrial membrane potential

    • Reactive oxygen species production

• Therapeutic intervention assessment:

  • Use MRPS34 antibodies to evaluate the efficacy of interventions targeting age-related mitochondrial dysfunction

  • Monitor changes in MRPS34 levels and mitochondrial function in response to treatments

The progressive nature of MRPS34-related defects makes this protein a potentially valuable biomarker for tracking age-related mitochondrial dysfunction and evaluating interventions.

What is the relationship between MRPS34 and respiratory chain complex assembly?

The relationship between MRPS34 and respiratory chain complex assembly has been elucidated through research on MRPS34 mutant models:

Molecular pathway from MRPS34 to respiratory chain assembly:

• MRPS34 deficiency impacts mitochondrial translation:

  • Compromised MRPS34 function leads to decreased stability of the small ribosomal subunit

  • This results in reduced synthesis of all 13 mitochondrially-encoded polypeptides

• Reduced mitochondrially-encoded subunits affect complex assembly:

  • Complexes I, III, IV, and V contain mitochondrially-encoded subunits

  • Deficiency in these subunits leads to incomplete complex assembly

  • BN-PAGE analysis has confirmed reduced levels of assembled respiratory complexes in MRPS34 mutant tissues

• Secondary effects on nuclear-encoded subunits:

  • In aged MRPS34 mutant mice, levels of nuclear-encoded mitochondrial proteins (NDUFA9, COXIV) are also reduced

  • This suggests a retrograde response to the decreased levels of mitochondrially-encoded proteins

• Tissue-specific patterns of complex deficiencies:

  • Heart: Primary reduction in Complexes I and IV

  • Liver: Reduction in Complexes I, III, IV, and V

  • These patterns may reflect tissue-specific energy demands and mitochondrial composition

Understanding this relationship provides insights into how defects in mitochondrial ribosomal proteins can lead to broad mitochondrial dysfunction through impaired respiratory chain complex assembly.

How can quantitative analyses of MRPS34 be used to assess mitochondrial ribosome integrity?

Quantitative analysis of MRPS34 using antibody-based methods can serve as a powerful approach to assess mitochondrial ribosome integrity:

Methodological approaches:

• Density gradient analysis with immunoblotting:

  • Separate mitochondrial ribosomal subunits and assembled ribosomes using sucrose gradient centrifugation

  • Analyze fractions by Western blotting using MRPS34 antibodies

  • Quantify the distribution of MRPS34 across gradient fractions to assess ribosomal assembly state

• Co-immunoprecipitation studies:

  • Use MRPS34 antibodies to pull down associated ribosomal components

  • Analyze the composition of immunoprecipitates to assess ribosome integrity

  • Compare results between normal and pathological conditions

• Proximity labeling approaches:

  • Combine MRPS34 antibodies with proximity labeling techniques (BioID, APEX)

  • Identify proteins in close proximity to MRPS34 in intact mitochondria

  • Map the mitochondrial ribosome interactome in different physiological states

• Quantitative mass spectrometry:

  • Use MRPS34 antibodies for immunoprecipitation followed by mass spectrometry

  • Determine the stoichiometry of mitochondrial ribosome components

  • Identify potential post-translational modifications that affect ribosome assembly

These approaches can provide insights into how mitochondrial ribosome integrity is affected in various pathological conditions, including mitochondrial diseases, neurodegenerative disorders, and aging.

What experimental designs can distinguish between primary MRPS34 defects and secondary mitochondrial dysfunction?

Distinguishing between primary MRPS34 defects and secondary mitochondrial dysfunction requires carefully designed experimental approaches:

Strategic experimental designs:

• Temporal analysis of molecular events:

  • Use time-course experiments to establish the sequence of molecular changes

  • In MRPS34 primary defects, changes in MRPS34 levels and 12S rRNA stability should precede broader mitochondrial dysfunction

  • Monitor using MRPS34 antibodies in combination with functional assays

• Genetic rescue experiments:

  • Re-express wild-type MRPS34 in deficient cells/tissues

  • Analyze whether this reverses observed mitochondrial defects

  • A complete or substantial rescue would suggest a primary MRPS34 defect

• Structure-function correlations:

  • Generate MRPS34 variants with specific mutations

  • Assess their impact on ribosome assembly and mitochondrial translation

  • Correlate molecular defects with functional consequences

• Multi-omics integration:

  • Combine proteomics (using MRPS34 antibodies), transcriptomics, and metabolomics

  • Compare patterns of molecular changes with known signatures of primary MRPS34 defects

  • Use computational modeling to distinguish primary from secondary effects

• In vivo models with tissue-specific manipulation:

  • Generate tissue-specific MRPS34 knockout or knockdown models

  • Compare the resulting phenotypes with those observed in disease states

  • Analyze tissue-specific responses using MRPS34 antibodies and functional assays

These experimental approaches can help researchers accurately diagnose the role of MRPS34 in mitochondrial disorders and develop targeted therapeutic strategies.

How can MRPS34 antibodies be used to investigate potential therapeutic targets for mitochondrial diseases?

MRPS34 antibodies can be valuable tools for investigating potential therapeutic targets for mitochondrial diseases through several sophisticated research applications:

Therapeutic target identification and validation:

• High-throughput screening approaches:

  • Use MRPS34 antibodies in cell-based assays to screen for compounds that stabilize MRPS34 or enhance its function

  • Develop immunofluorescence-based screening assays to identify molecules that restore mitochondrial ribosome assembly

• Target engagement studies:

  • Utilize MRPS34 antibodies to confirm that candidate therapeutic molecules physically interact with their intended targets

  • Employ cellular thermal shift assays (CETSA) with MRPS34 antibodies to verify binding of compounds to MRPS34 in cells

• Therapeutic efficacy assessment:

  • Monitor changes in MRPS34 levels and mitochondrial ribosome assembly in response to treatment

  • Correlate these changes with improvements in mitochondrial function and disease phenotypes

  • Experimental data from MRPS34 mutant mice suggest that enhancing mitochondrial translation could be beneficial in mitochondrial disorders

• Biomarker development:

  • Use MRPS34 antibodies to develop assays for monitoring disease progression and treatment response

  • Quantify MRPS34 and associated proteins in accessible samples (e.g., blood cells, skin fibroblasts)

  • Establish correlations between MRPS34-related parameters and clinical outcomes

• Combination therapy evaluation:

  • Assess how interventions targeting MRPS34 or mitochondrial translation combine with other mitochondrial therapies

  • Use MRPS34 antibodies to elucidate mechanisms of synergy or antagonism

Through these approaches, MRPS34 antibodies can contribute to the development of novel therapeutic strategies for mitochondrial diseases, a group of disorders that currently have limited treatment options.