MRPS18A Antibody

What is MRPS18A and what is its primary cellular function?

MRPS18A (Mitochondrial Ribosomal Protein S18A) is a nuclear-encoded protein that is essential for protein synthesis within the mitochondrion. It belongs to the ribosomal protein S18P family and is a component of the small 28S subunit of mitochondrial ribosomes (mitoribosomes) . Mammalian mitoribosomes differ significantly from their prokaryotic counterparts, having an estimated 75% protein to rRNA composition (reversed ratio compared to prokaryotic ribosomes) . MRPS18A is one of three human mitochondrial proteins that show significant sequence similarity to bacterial S18 proteins, though interestingly, the primary sequences of these three human mitochondrial S18 proteins (MRPS18A, MRPS18B, MRPS18C) are no more closely related to each other than they are to prokaryotic S18 proteins . This suggests distinct evolutionary paths and potentially specialized functions for each variant.

How does MRPS18A expression differ between normal and cancer cells?

Research has identified MRPS18A as being upregulated in breast cancer cells compared to normal breast cells . This upregulation is not homogeneous across all cancer cells, suggesting the existence of sub-populations within tumors with varying expression levels . The increased expression of MRPS18A in cancer cells is primarily explained by the enhanced energy metabolism characteristic of rapidly proliferating cancer cells . This altered energy metabolism is considered one of the hallmarks of cancer, with mitochondria playing a central role in this process . The differential expression makes MRPS18A a potential biomarker for certain cancers and could open new diagnostic and therapeutic avenues .

What are the optimal applications for MRPS18A antibodies in research?

MRPS18A antibodies have been validated primarily for Western Blot (WB) and ELISA applications, with demonstrated reactivity in human samples . For Western Blot applications, recommended dilutions typically range from 1:5000 to 1:50000, though optimal dilutions should be determined for each experimental system . MRPS18A antibodies have shown positive Western Blot results in multiple human cell lines including HepG2, A549, HeLa, L02, and MCF-7 cells .

When designing experiments using MRPS18A antibodies, it's important to note that some antibodies may recognize conformational epitopes rather than linear ones, which can affect their performance in different applications. For example, in one study, an antibody designated BC5 recognized MRPS18A in far-western blot analysis but not in standard western blot, indicating recognition of a conformational epitope .

What is the discrepancy between calculated and observed molecular weights of MRPS18A, and how does this affect antibody selection?

An interesting characteristic of MRPS18A is the difference between its calculated molecular weight of 22 kDa (based on its 196 amino acid sequence) and its observed molecular weight of approximately 15 kDa in experimental conditions . This discrepancy is important to consider when selecting antibodies and interpreting experimental results.

When performing Western Blot analysis, researchers should expect to observe bands at approximately 15 kDa rather than at the calculated 22 kDa position. Additionally, when analyzing mitochondrial fractions, multiple bands may be observed, with some potentially representing degradation products . For instance, far-western blot analysis of MCF7 cell extracts has shown three bands in whole cell extracts and two in the mitochondrial fraction, with the upper band corresponding to MRPS18A . When selecting antibodies, researchers should verify that the antibody can recognize the protein at its observed molecular weight rather than just its theoretical weight.

How should researchers design experiments to study MRPS18A expression differences between normal and cancer tissues?

When designing experiments to study differential expression of MRPS18A between normal and cancer tissues, researchers should consider a multi-faceted approach:

• Cell and Tissue Selection: Include multiple cancer cell lines (such as MCF-7 for breast cancer) alongside normal cell counterparts. For tissue analysis, paired samples of tumor and adjacent normal tissue from the same patients provide the most controlled comparison .

• Subcellular Fractionation: Since MRPS18A is a mitochondrial protein, proper subcellular fractionation to isolate mitochondria is crucial. Protocols should separate whole cell extracts, cytoplasmic fractions, and mitochondrial fractions for comprehensive analysis .

• Detection Methods:

  • Western Blot: Use optimized antibody dilutions (1:5000-1:50000) with appropriate controls

  • Immunohistochemistry (IHC): For tissue sections to visualize spatial distribution

  • qRT-PCR: To measure mRNA expression levels

  • Proteomics: Mass spectrometry-based approaches for unbiased protein quantification

• Analysis of Cell Subpopulations: Since MRPS18A upregulation is not homogeneous in all cancer cells, consider single-cell analysis techniques or sorting of cell subpopulations (e.g., based on stem cell markers or other cancer markers like cytokeratin 19 and cytokeratin 14) .

• Functional Studies: Couple expression analysis with functional studies, such as knockdown/overexpression experiments, to assess the biological significance of expression differences.

What controls are essential when using MRPS18A antibodies in experimental procedures?

When using MRPS18A antibodies, the following controls are essential:

• Positive Controls: Include cell lines known to express MRPS18A, such as HepG2, A549, HeLa, L02, or MCF-7 cells .

• Negative Controls:

  • Primary antibody controls: Omit the primary MRPS18A antibody while keeping all other reagents

  • Isotype controls: Use matching isotype IgG from the same host species (rabbit IgG for rabbit-derived MRPS18A antibodies)

  • MRPS18A-knockdown samples: When possible, include samples where MRPS18A has been knocked down using siRNA or CRISPR

• Specificity Controls:

  • Peptide competition assays: Pre-incubate antibody with excess MRPS18A recombinant protein or peptide

  • Alternative antibodies: Use antibodies recognizing different epitopes of MRPS18A

  • Include unrelated antibodies of the same scaffold as a control (e.g., in one study, BC6 antibody was used as a control to confirm that binding was specific to the antigen-binding region of the BC5 antibody)

• Loading Controls: For Western Blots, include appropriate loading controls:

  • Total protein normalization methods

  • Housekeeping proteins appropriate for mitochondrial proteins (such as VDAC for mitochondrial fractions)

• Subcellular Fractionation Controls: When analyzing mitochondrial fractions, include markers for mitochondria (e.g., COX IV) and for other cellular compartments (e.g., GAPDH for cytoplasm, Lamin A/C for nucleus) to verify fractionation quality.

How should researchers interpret contradictory data on MRPS18A localization within mitochondrial ribosomes?

When faced with such contradictions, researchers should:

What metrics should be used to quantify MRPS18A expression differences between normal and cancer tissues?

For rigorous quantification of MRPS18A expression differences, researchers should consider multiple metrics:

• Fold change: Calculate the ratio of expression in cancer vs. normal tissues, using appropriate normalization methods.

• Statistical significance: Employ appropriate statistical tests (t-test, ANOVA, or non-parametric alternatives) with correction for multiple testing when applicable.

• Effect size: Report measures like Cohen's d to quantify the magnitude of differences independent of sample size.

• Heterogeneity measures: Since MRPS18A expression is not uniform across all cancer cells , quantify the variance or distribution of expression within samples.

• Correlation metrics: Assess how MRPS18A expression correlates with:

  • Clinical parameters (stage, grade, patient survival)

  • Expression of other mitochondrial proteins

  • Metabolic markers

  • Proliferation indices

• Multivariate analyses: Use principal component analysis or clustering to position MRPS18A expression within broader molecular profiles.

• ROC curve analysis: For biomarker potential assessment, calculate sensitivity, specificity, and area under the curve.

For publication-quality analysis, present data in tables with multiple metrics rather than simply reporting p-values, and include visualizations that capture both the magnitude of differences and the variability within groups.

What are the most common issues when detecting MRPS18A in Western Blot experiments and how can they be resolved?

Common issues when detecting MRPS18A by Western Blot include:

• Incorrect band size: The observed molecular weight of MRPS18A (15 kDa) differs from its calculated weight (22 kDa) .

  • Solution: Always confirm the expected observed size for your specific antibody through product datasheets or literature.

• Multiple bands: Far-western blot analysis of cell extracts has shown multiple bands, with some potentially representing degradation products .

  • Solution: Include proper positive controls and subcellular fractionation to identify which band represents the intact protein. Consider using freshly prepared samples with protease inhibitors to minimize degradation.

• Conformational epitope recognition: Some antibodies (like BC5) recognize conformational rather than linear epitopes of MRPS18A .

  • Solution: Try different denaturation conditions, native gels, or far-western blotting techniques that may preserve certain protein conformations.

• Weak signal: This may occur due to low abundance of MRPS18A in some cell types.

  • Solution: Optimize antibody concentration (try the recommended range of 1:5000-1:50000) , increase protein loading, use enhanced chemiluminescence detection systems, or consider enriching for mitochondrial fractions.

• High background: Non-specific binding can obscure specific signals.

  • Solution: Optimize blocking conditions (try different blocking agents), increase washing steps, and adjust antibody dilution. Consider using recombinant antibodies, which often have higher specificity .

• Inconsistent results across cell lines: Expression levels vary between cell types.

  • Solution: Begin with validated positive control cell lines such as HepG2, A549, HeLa, L02, or MCF-7 cells .

How can researchers effectively distinguish between the three MRPS18 variants (MRPS18A, MRPS18B, MRPS18C) in experimental settings?

Distinguishing between the three MRPS18 variants requires careful experimental design:

• Antibody selection: Choose antibodies with validated specificity for each variant. Since the three MRPS18 proteins have only 25-30% sequence identity to each other , carefully selected antibodies should be specific. Verify antibody specificity using:

  • Recombinant proteins for each variant

  • Cells overexpressing specific variants

  • Samples with knockdown of specific variants

• Molecular techniques:

  • RT-qPCR: Design primers specific to unique regions of each variant's mRNA

  • Mass spectrometry: Identify variant-specific peptides for unambiguous identification

  • CRISPR knockout/knockdown: Generate cells lacking specific variants to serve as negative controls

• Subcellular localization analysis: The variants may have subtly different localizations within mitochondria or cells:

  • High-resolution microscopy with variant-specific antibodies

  • Subcellular fractionation followed by Western blotting

  • Proximity labeling approaches to identify differential interaction partners

• Functional discrimination: Design assays that exploit functional differences between variants:

  • MRPS18B has been linked to immortalization when overexpressed

  • Look for variant-specific interactions with regulatory proteins

• Expression pattern analysis: The variants may be differentially expressed across tissues or cellular conditions . Comparing expression patterns can help validate the specificity of detection methods.

• Recombinant protein standards: Include titrated amounts of recombinant proteins for each variant as standards for quantitative assays.

How might MRPS18A be leveraged as a cancer biomarker, and what validation studies would be necessary?

Based on its upregulation in breast cancer cells , MRPS18A shows potential as a cancer biomarker. To develop and validate MRPS18A as a clinically useful biomarker, researchers should pursue:

• Large-scale expression studies:

  • Analyze MRPS18A expression across diverse cancer types and subtypes

  • Include adequate sample sizes with matched normal tissues

  • Assess correlation with existing biomarkers and clinical parameters

• Multimodal detection standardization:

  • Develop standardized IHC protocols with scoring systems

  • Validate antibodies across multiple platforms (IHC, ELISA, Western blot)

  • Establish quantitative PCR protocols for transcript detection

• Prognostic value assessment:

  • Perform survival analyses correlating MRPS18A expression with patient outcomes

  • Conduct multivariate analyses to determine independent prognostic value

  • Evaluate utility in specific clinical contexts (early detection, treatment response)

• Functional validation:

  • Determine whether MRPS18A upregulation is a driver or passenger in cancer progression

  • Investigate downstream effects of MRPS18A modulation

  • Assess relationship to cancer hallmarks beyond altered metabolism

• Combinatorial biomarker panels:

  • Evaluate if MRPS18A adds value to existing biomarker panels

  • Develop optimized algorithms incorporating MRPS18A with other markers

  • Test performance in blinded validation cohorts

• Liquid biopsy potential:

  • Investigate whether MRPS18A protein or transcripts can be detected in bodily fluids

  • Assess correlation between tissue and liquid biopsy measurements

• Technology transfer:

  • Develop assays suitable for clinical laboratory implementation

  • Conduct comparative studies with existing diagnostic methods

What is the relationship between MRPS18A and other mitochondrial ribosomal proteins in cancer progression, and how should this be studied?

Understanding the relationship between MRPS18A and other mitochondrial ribosomal proteins (MRPs) in cancer requires sophisticated experimental approaches:

• Comprehensive expression profiling:

  • Analyze co-expression patterns of multiple MRPs across cancer types

  • Identify coordinated expression changes and potential regulatory mechanisms

  • Determine whether MRPS18A upregulation occurs in isolation or as part of broader mitochondrial ribosome remodeling

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with MRPS18A to identify direct interactors

  • Use proximity labeling methods (BioID, APEX) to map the broader interaction network

  • Compare interaction networks between normal and cancer cells

• Functional redundancy assessment:

  • Conduct simultaneous and sequential knockdown of MRPS18A and other MRPs

  • Evaluate compensatory mechanisms and synthetic lethality relationships

  • Determine if MRPS18A has unique functions not shared by other family members

• Mitoribosome assembly studies:

  • Investigate how MRPS18A and other MRPs contribute to mitoribosome assembly in cancer cells

  • Assess whether cancer-specific alterations in mitoribosome composition occur

  • Determine if MRPS18A is rate-limiting for mitoribosome formation

• Metabolic impact analysis:

  • Measure how modulation of MRPS18A and other MRPs affects mitochondrial translation

  • Assess consequences for oxidative phosphorylation and metabolic adaptation

  • Investigate whether targeting specific MRPs can reverse the Warburg effect

• Extra-ribosomal function exploration:

  • Investigate potential moonlighting functions of MRPS18A outside the mitoribosome

  • Compare with known extra-ribosomal functions of other MRPs (e.g., MRPS29's role in apoptosis control )

  • Determine if these functions contribute to cancer progression

• Therapeutic targeting strategies:

  • Evaluate whether MRPS18A represents a vulnerability in cancer cells

  • Compare with therapeutic potential of targeting other MRPs

  • Develop approaches to selectively disrupt cancer-specific mitoribosome functions

This research direction would benefit from integrating computational approaches with experimental validation, potentially revealing new therapeutic targets within the mitochondrial translation machinery.