MRPS22 Antibody

What is MRPS22 and what is its biological significance?

MRPS22 (Mitochondrial Ribosomal Protein S22) is a 360-amino-acid protein that functions as a key component of the mitochondrial ribosome small subunit (28S), which comprises a 12S rRNA and approximately 30 distinct proteins . This protein plays a crucial role in mitochondrial protein synthesis and is essential for normal embryonic development. MRPS22 is also known by several alternative names including C3orf5, RPMS22, GK002, MRP-S22, and S22mt . The protein has a calculated molecular weight of 41 kDa, though it typically appears at approximately 38 kDa on Western blots due to post-translational modifications or protein folding characteristics . Mutations in the MRPS22 gene have been linked to combined oxidative phosphorylation deficiency type 5 (COXPD5) and, more recently, to premature ovarian insufficiency (POI) in adolescents .

What types of MRPS22 antibodies are available for research applications?

Several types of MRPS22 antibodies are commercially available for research purposes, including:

These antibodies differ in their immunogens, with some targeting specific peptide regions (e.g., aa 50-200 of human MRPS22) and others using fusion proteins or full-length proteins as immunogens . The selection of the appropriate antibody depends on the specific experimental requirements, target species, and intended application.

What are the optimal conditions for using MRPS22 antibodies in Western blotting?

For Western blotting applications with MRPS22 antibodies, several technical considerations are important:

• Dilution Range: The recommended dilution range for Western blot applications is typically 1:200-1:1000, though this may vary by manufacturer . For example, Abcam's ab224341 has been validated at 1:100 dilution .

• Sample Preparation: MRPS22 antibodies have been validated with various cell and tissue lysates including:

  • Human cell lines: Jurkat cells, RT4, U-251 MG, U-2 OS

  • Mouse tissues: liver, spleen

  • Rat tissues: spleen

  • Mouse cell lines: NIH/3T3

• Expected Molecular Weight: Researchers should expect to observe a band at approximately 38-41 kDa, with the calculated molecular weight being 41 kDa and the observed weight often closer to 38 kDa .

• Controls: Positive controls using tissues known to express MRPS22 (such as Jurkat cells) are recommended to validate antibody performance in each experimental system .

• Titration: It is recommended that researchers titrate the antibody in each specific testing system to obtain optimal results, as sample type and preparation method can influence antibody performance .

What methodological approaches are recommended for immunohistochemistry with MRPS22 antibodies?

For immunohistochemistry applications using MRPS22 antibodies:

• Dilution Range: The recommended dilution range for IHC applications is typically 1:20-1:200 , with specific antibodies like Abcam's ab224341 validated at 1:200 dilution .

• Antigen Retrieval: For optimal results, antigen retrieval with TE buffer pH 9.0 is suggested. Alternatively, citrate buffer pH 6.0 may be used depending on the specific tissue and fixation method .

• Validated Tissues: MRPS22 antibodies have been successfully used in IHC for:

  • Human breast cancer tissue

  • Human lymph node tissue

  • Other human tissues where mitochondrial function is being studied

• Detection Systems: Standard secondary antibody detection systems compatible with rabbit IgG are appropriate, with careful consideration of background control in highly metabolic tissues .

How can MRPS22 antibodies be used to investigate mitochondrial dysfunction in disease models?

MRPS22 antibodies provide valuable tools for investigating mitochondrial dysfunction in various disease models:

• Expression Analysis: Quantification of MRPS22 protein levels using Western blotting can help assess mitochondrial ribosome integrity in disease models . This is particularly relevant in tissues with high energy demands.

• Localization Studies: Using immunofluorescence or immunohistochemistry, researchers can examine subcellular localization of MRPS22 and potential alterations in mitochondrial distribution in diseased tissues .

• Co-immunoprecipitation: MRPS22 antibodies can be used for immunoprecipitation (IP) at recommended concentrations of 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate to study protein-protein interactions within the mitochondrial ribosome .

• Disease Models: Research has shown that mutations in MRPS22 are associated with combined oxidative phosphorylation deficiency type 5 (COXPD5) and premature ovarian insufficiency (POI) . MRPS22 antibodies can be used to investigate these disease models.

• Comparative Studies: Comparing MRPS22 expression between control and patient-derived fibroblasts or in animal models can reveal insights into mitochondrial dysfunction mechanisms .

What approaches can be used to validate specificity when working with MRPS22 antibodies?

Validating antibody specificity is crucial for generating reliable research data. For MRPS22 antibodies, consider these approaches:

• Knockout/Knockdown Controls: While complete knockout of MRPS22 in mice results in embryonic lethality , RNAi-mediated knockdown in cell lines can serve as negative controls for antibody validation.

• Multiple Antibody Approach: Using different antibodies that recognize distinct epitopes of MRPS22 can provide confirmatory evidence of specificity .

• Peptide Competition Assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals in Western blot or immunohistochemistry.

• Cross-Reactivity Testing: Verify species cross-reactivity experimentally even when predicted by sequence homology, particularly when working with novel model organisms .

• Signal Correlation: Correlation between protein and mRNA expression levels can provide additional evidence for antibody specificity .

How should researchers address inconsistent MRPS22 antibody performance across different applications?

When facing inconsistent results with MRPS22 antibodies:

• Application-Specific Optimization: Different applications require different antibody concentrations. For example:

  • WB: 1:200-1:1000

  • IHC: 1:20-1:200

  • IP: 0.5-4.0 μg for 1.0-3.0 mg of total protein

• Sample Preparation Considerations: Mitochondrial proteins may require specific lysis conditions to preserve native conformation. Consider using mitochondrial isolation protocols before standard lysis if signal is weak.

• Buffer Compatibility: The storage buffer for many MRPS22 antibodies contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Ensure experimental buffers are compatible with these components.

• Sensitivity Analysis: If signal is absent, consider using more sensitive detection systems or increasing protein loading. Conversely, if background is high, optimize blocking conditions and increase antibody dilution.

• Batch Variation: Different lots of the same antibody may show performance variability. Validation with positive controls before experimental use is recommended .

What considerations are important when interpreting MRPS22 expression data from patient samples?

When analyzing MRPS22 expression in patient samples:

• Normal Variation: Consider that even in disease states, MRPS22 expression or protein levels might not always show detectable changes in all tissue types. For example, studies of patient-derived fibroblasts with MRPS22 mutations showed no detectable changes in protein or mRNA expression levels compared to controls .

• Tissue-Specific Effects: MRPS22 mutations may have tissue-specific effects. For instance, mutations that cause POI may not affect mitochondrial function in fibroblasts but critically impact ovarian tissue .

• Functional Redundancy: Consider that functional studies may be necessary to complement expression data, as normal expression levels do not rule out functional defects in the protein.

• Correlation with Disease Markers: Analyze MRPS22 expression in relation to other mitochondrial markers or disease-specific parameters for more meaningful interpretation.

• Mutation Context: Different mutations in MRPS22 may have varying effects on protein function without altering expression levels. The p.R202H variant, for example, did not affect expression but was associated with disease .

What experimental approaches are recommended for studying MRPS22 in relation to mitochondrial translation?

To study MRPS22's role in mitochondrial translation:

What are the key considerations when designing experiments to investigate MRPS22 mutations in disease models?

When investigating MRPS22 mutations in disease contexts:

• Mutation Selection: Consider that different mutations may have varying effects. Known pathogenic variants include c.404G>A (p.R135Q) and c.605G>A (p.R202H) .

• Tissue Relevance: While fibroblasts are commonly used for accessibility, they may not reflect the tissue-specific effects of MRPS22 mutations. Consider using disease-relevant tissues or differentiated cells when possible .

• Functional Assays: Include comprehensive functional assessments:

  • Protein expression and stability

  • Mitochondrial ribosome assembly

  • Mitochondrial translation efficiency

  • Oxidative phosphorylation activity

  • Cell viability and growth parameters

• Controls Selection: Use appropriate controls including:

  • Wild-type cells/tissues

  • Heterozygous mutation carriers

  • Cells with mutations in other mitochondrial ribosomal proteins for comparison

• Rescue Experiments: Consider complementation with wild-type MRPS22 to confirm the causative role of identified mutations .

How might MRPS22 antibodies contribute to understanding tissue-specific mitochondrial dysfunction?

MRPS22 antibodies can advance understanding of tissue-specific mitochondrial dysfunction through:

• Tissue Expression Profiling: Systematic analysis of MRPS22 expression across different tissues can provide insights into tissue-specific vulnerabilities to mitochondrial dysfunction .

• Developmental Studies: Investigating MRPS22 expression during development may explain why certain mutations cause embryonic lethality in mouse models while causing specific phenotypes like POI in humans .

• Cell Type-Specific Analysis: IHC and IF applications of MRPS22 antibodies enable assessment of expression in specific cell types within heterogeneous tissues, potentially revealing cell-specific vulnerabilities .

• Disease Model Comparisons: Using MRPS22 antibodies to compare expression and localization across different disease models can help identify common pathways in mitochondrial dysfunction .

• Proximity Labeling Approaches: Combining MRPS22 antibodies with proximity labeling techniques can map tissue-specific protein interaction networks around mitochondrial ribosomes.

What techniques can be employed to study MRPS22's role in mitochondrial disease pathogenesis?

Advanced techniques for studying MRPS22's role in disease pathogenesis include:

• Patient-Derived Models: Using patient-derived fibroblasts, induced pluripotent stem cells (iPSCs), or differentiated cells to study the effects of MRPS22 mutations in relevant cellular contexts .

• CRISPR/Cas9 Genome Editing: Generation of isogenic cell lines with specific MRPS22 mutations to study their functional consequences without confounding genetic background effects.

• Tissue-Specific Conditional Knockouts: Since complete knockout is embryonic lethal, tissue-specific or inducible knockout models can provide insights into tissue-specific requirements for MRPS22 .

• Multi-omics Approaches: Integration of proteomics, transcriptomics, and metabolomics data to comprehensively characterize the consequences of MRPS22 dysfunction.

• Super-Resolution Microscopy: Combining MRPS22 antibodies with super-resolution microscopy techniques to visualize mitochondrial ribosome organization and potential alterations in disease states .