MRPS10 Antibody

What is MRPS10 and why is it relevant for mitochondrial research?

MRPS10 (Mitochondrial Ribosomal Protein S10) is a nuclear-encoded component of the small 28S subunit of mitochondrial ribosomes. It plays a crucial role in protein synthesis within the mitochondrion . The mitochondrial ribosome (mitoribosome) differs significantly from cytosolic ribosomes, containing approximately 75% protein to rRNA composition compared to prokaryotic ribosomes where this ratio is reversed . MRPS10 belongs to the ribosomal protein S10P family and is essential for mitochondrial translation. Research on MRPS10 can provide insights into mitochondrial function, protein synthesis disorders, and related pathologies.

How should MRPS10 antibody validation be performed when studying novel cell lines or tissues?

When validating MRPS10 antibodies in novel cell lines or tissues, a multi-step approach is recommended:

• Positive control selection: Use validated positive controls such as HeLa cells, human liver tissue, HepG2 cells, or human brain tissue, which have been demonstrated to express MRPS10 .

• Western blot validation: First, confirm antibody specificity through Western blot analysis, looking for a distinct band at approximately 23 kDa, which is the observed molecular weight of MRPS10 .

• Dilution optimization: Test a range of antibody dilutions (e.g., 1:500, 1:1000, 1:3000 for WB) to determine optimal signal-to-noise ratio .

• Negative controls: Include appropriate negative controls, such as:

  • Secondary antibody only

  • Isotype control antibody

  • Tissues known not to express the target

• Cross-reactivity assessment: If working with non-human samples, consider the degree of sequence homology (e.g., mouse MRPS10 has 88% sequence identity with human, bovine has 93%) .

• Subcellular localization confirmation: Given MRPS10's mitochondrial localization, immunofluorescence co-staining with established mitochondrial markers can provide additional validation .

How can researchers troubleshoot inconsistent MRPS10 antibody performance across different experimental systems?

Inconsistent antibody performance can result from multiple factors. When troubleshooting MRPS10 antibody performance issues:

• Epitope accessibility: The accessibility of the MRPS10 epitope may vary depending on sample preparation methods. Different antibodies target different regions of MRPS10, which can affect accessibility:

  • N-terminal region antibodies (e.g., GTX119522) target the N1C3 region

  • Central region antibodies target sequences like TAFGAVCRRLWQGLGN...

  • Full-length protein antibodies may recognize different epitopes

• Sample preparation considerations:

  • For Western blotting: Complete protein denaturation is essential; insufficient SDS or reducing agent can affect epitope exposure

  • For IHC/ICC: Antigen retrieval methods should be optimized; excessive fixation can mask epitopes

• Cross-reactivity analysis: Test for potential cross-reactivity with other proteins, particularly other mitochondrial ribosomal proteins that may share sequence homology .

• Antibody selection based on application: Some antibodies perform better in certain applications than others:

  • For quantitative Western blotting, affinity-purified antibodies typically yield more consistent results

  • For immunohistochemistry, antibodies validated specifically for this application should be selected

• Positive control inclusion: Always include a known positive control (e.g., HeLa cells) alongside experimental samples to verify antibody functionality within each experiment .

What are the critical factors to consider when using MRPS10 antibodies for co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments with MRPS10 antibodies, several factors should be considered:

• Antibody binding characteristics: Select antibodies with demonstrated affinity purification against MRPS10 recombinant protein . This increases the likelihood of successful immunoprecipitation.

• Buffer composition: The composition of lysis and wash buffers is critical:

  • For mitochondrial proteins, specialized lysis buffers may be required to effectively solubilize mitochondrial membranes

  • Consider nonionic detergents (e.g., NP-40 or Triton X-100) at 0.5-1% to maintain protein-protein interactions

  • Salt concentration should be optimized (typically 100-150 mM NaCl) to minimize nonspecific binding while preserving specific interactions

• Crosslinking considerations: For transient or weak interactions, chemical crosslinking prior to lysis may be necessary. Common crosslinkers include formaldehyde (0.1-1%) or DSP (dithiobis[succinimidyl propionate]) .

• Control experiments:

  • Negative control using non-specific IgG from the same species as the MRPS10 antibody

  • Reverse co-IP validation (immunoprecipitate with antibodies against suspected interaction partners)

  • Input control (5-10% of lysate used for co-IP)

• Detection strategy: For Western blot detection after co-IP:

  • Use the recommended dilution for the MRPS10 antibody (typically 1:500-1:3000)

  • Consider using antibodies raised in different species for the co-IP and detection steps to avoid detection of the immunoprecipitating antibody

How can MRPS10 antibodies be utilized in mitochondrial dysfunction research?

MRPS10 antibodies can serve as valuable tools in studying mitochondrial dysfunction:

• Mitochondrial ribosome assembly assessment: Changes in MRPS10 levels or localization can indicate defects in mitochondrial ribosome assembly. Western blot analysis using MRPS10 antibodies can quantify these changes .

• Mitochondrial translation evaluation: Since MRPS10 is essential for mitochondrial translation, monitoring its expression can provide insights into translational defects. Comparative analysis of MRPS10 levels across different conditions can indicate translational stress .

• Disease model investigation: MRPS10 antibodies can be used to study mitochondrial ribosomal protein expression in disease models associated with mitochondrial dysfunction, including:

  • Neurodegenerative disorders

  • Metabolic diseases

  • Cancer models with altered mitochondrial function

• Protein-protein interaction studies: Co-immunoprecipitation with MRPS10 antibodies can identify novel interaction partners within the mitochondrial translation machinery .

• Tissue-specific expression analysis: Immunohistochemistry using MRPS10 antibodies can reveal tissue-specific differences in mitochondrial ribosome composition and potential dysfunction .

What considerations should be made when interpreting MRPS10 antibody results in the context of autoimmune disease research?

Recent research has identified autoantibodies against various intracellular proteins, including mitochondrial components, in autoimmune conditions. When investigating MRPS10 in autoimmune contexts:

• Background signal differentiation: Distinguish between specific MRPS10 signal and background that may result from autoantibodies in patient samples binding to secondary antibodies .

• Proteome-wide autoantibody screening (PWAbS): Recent studies have employed techniques like PWAbS to identify disease-specific autoantibody signatures. When MRPS10 is detected in such screenings, verification using purified antibodies is essential .

• Cross-reactivity analysis: Patient autoantibodies may recognize epitopes that cross-react with MRPS10. Competitive binding assays with purified MRPS10 antibodies can help determine specificity .

• Control selection: Appropriate controls are critical when analyzing MRPS10 in autoimmune contexts:

  • Healthy control samples

  • Disease-specific controls (e.g., samples from patients with different autoimmune conditions)

  • Technical controls to rule out non-specific binding

• Longitudinal sample analysis: As demonstrated in COVID-19 research, autoantibody profiles may change over the course of disease. Temporal analysis using MRPS10 antibodies can provide insights into disease progression .

How can MRPS10 antibodies contribute to understanding mitochondrial-nuclear communication?

MRPS10 antibodies can provide insights into mitochondrial-nuclear communication mechanisms:

• Subcellular localization studies: Immunofluorescence with MRPS10 antibodies can track potential dual localization of this protein under different cellular conditions .

• Stress response analysis: During mitochondrial stress, communication pathways between mitochondria and nucleus are activated. MRPS10 antibodies can be used to monitor changes in MRPS10 expression and localization during:

  • Oxidative stress

  • mtDNA depletion

  • Mitochondrial protein import defects

• Protein complex analysis: Combining MRPS10 antibodies with antibodies against nuclear-encoded transcription factors in co-immunoprecipitation studies can help identify novel protein complexes involved in mitochondrial-nuclear signaling .

• Chromatin immunoprecipitation (ChIP) applications: For potential nuclear-localized MRPS10, ChIP using MRPS10 antibodies could identify DNA binding sites and potential transcriptional regulatory roles.

• Proximity ligation assays: Using MRPS10 antibodies in conjunction with antibodies against nuclear proteins in proximity ligation assays can visualize and quantify interactions at the mitochondrial-nuclear interface.

What are the considerations for using MRPS10 antibodies in studying mitochondrial dynamics during cell differentiation?

Mitochondrial ribosomes undergo significant remodeling during cellular differentiation processes. When using MRPS10 antibodies to study these dynamics:

• Temporal expression pattern analysis: Use MRPS10 antibodies for Western blot or immunofluorescence analysis at different time points during differentiation to track changes in expression levels and localization .

• Antibody selection for developmental studies: Consider antibodies targeting different epitopes of MRPS10 as protein conformations or interactions may change during differentiation:

  • N-terminal antibodies (e.g., GTX119522)

  • Central region antibodies (e.g., NBP1-83847)

• Multiplex immunofluorescence approaches: Combine MRPS10 antibodies with markers of:

  • Mitochondrial biogenesis (e.g., PGC-1α)

  • Mitochondrial fusion/fission proteins

  • Cell differentiation state markers

• Quantitative considerations: For accurate quantification during differentiation studies:

  • Use validated loading controls appropriate for mitochondrial proteins

  • Consider normalization to mitochondrial mass using mitochondrial markers

  • Account for potential changes in mitochondrial number and morphology

• Species-specific considerations: When studying differentiation in animal models, consider the species reactivity of MRPS10 antibodies:

  • Human, mouse, and rat reactivity is common for many MRPS10 antibodies

  • Species-specific optimization may be necessary for developmental studies

How can MRPS10 antibodies be integrated into high-throughput proteomics approaches?

Integrating MRPS10 antibodies into high-throughput proteomics offers several research advantages:

• Antibody arrays and multiplexed assays: MRPS10 antibodies can be included in antibody arrays for simultaneous detection of multiple mitochondrial proteins. When designing such arrays:

  • Validate antibody specificity in the array format

  • Optimize antibody concentrations to minimize cross-reactivity

  • Include appropriate controls for normalization

• Mass spectrometry immunoprecipitation: MRPS10 antibodies can be used for immunoprecipitation followed by mass spectrometry (IP-MS) to identify:

  • MRPS10 post-translational modifications

  • Protein interaction networks

  • Changes in protein complex composition under different conditions

• Single-cell proteomics applications: For single-cell analysis:

  • Validate antibody performance at the sensitivity required for single-cell detection

  • Consider signal amplification methods for low-abundance detection

  • Optimize fixation and permeabilization protocols for mitochondrial proteins

• Automation considerations: For automated high-throughput applications:

  • Standard antibody concentrations (typically 1:500-1:1000) may need adjustment

  • Consistent antibody lots are critical for reproducibility

  • Include inter-plate calibration standards

What are the considerations for using MRPS10 antibodies in conjunction with emerging spatial transcriptomics approaches?

Combining MRPS10 antibody-based protein detection with spatial transcriptomics requires careful methodological considerations:

• Dual protein-RNA detection protocols: When developing protocols for simultaneous detection:

  • Validate that RNA preservation steps do not interfere with MRPS10 epitope recognition

  • Optimize fixation conditions that preserve both protein epitopes and RNA integrity

  • Test potential RNase contamination in antibody preparations

• Sequential immunofluorescence and in situ hybridization:

  • Determine optimal antibody dilution for immunofluorescence (typically 1:100-1:1000)

  • Validate signal specificity in multiplexed detection systems

  • Develop appropriate controls for autofluorescence and non-specific binding

• Data integration approaches:

  • Develop registration methods to align protein and RNA signals

  • Consider computational approaches to correlate MRPS10 protein levels with mitochondrial transcript abundance

  • Account for differences in sensitivity between antibody-based detection and nucleic acid amplification

• Technical validation requirements:

  • Include spike-in controls for quantification

  • Perform replicate experiments to assess technical variability

  • Validate findings with orthogonal methods (e.g., Western blot, qPCR)

• Emerging applications: Consider using MRPS10 antibodies in cutting-edge approaches:

  • Spatial proteomics with multiplexed antibody staining

  • CODEX or other highly multiplexed imaging platforms

  • Integration with single-cell multi-omics approaches