MRPL38 Antibody

What is the molecular structure and function of MRPL38?

MRPL38 (also known as 39S ribosomal protein L38, mitochondrial, L38mt, or MRP-L38) is a 346 amino acid protein with a calculated molecular weight of approximately 41 kDa, though it is typically observed between 37-45 kDa in experimental conditions . As part of the phosphatidylethanolamine-binding protein family, it functions as a component of the large mitochondrial ribosomal subunit (39S), playing a crucial role in mitochondrial protein synthesis. In yeast models, studies have shown that high levels of mitochondrial large ribosomal subunit proteins help prevent loss of mitochondrial DNA in null mmf1 Saccharomyces cerevisiae cells, suggesting MRPL38 may have roles in maintaining mitochondrial genome integrity .

What species reactivity do commercially available MRPL38 antibodies demonstrate?

Based on extensive validation data, MRPL38 antibodies show reactivity with various species depending on the specific clone and manufacturer:

• Human, mouse, and rat reactivity is confirmed for several polyclonal antibodies

• Human and pig reactivity is documented for specific monoclonal antibodies

• Non-human primate reactivity has been reported for some antibodies

The cross-species reactivity suggests conservation of epitope regions across mammalian species, though researchers should verify reactivity for their specific experimental model .

What experimental applications are MRPL38 antibodies validated for?

MRPL38 antibodies have been rigorously validated for multiple applications:

Each application requires optimization for specific experimental conditions and sample types .

How should researchers optimize Western blot protocols for MRPL38 detection?

For optimal Western blot detection of MRPL38:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for tissue or cell lysis. For mitochondrial enrichment, consider subcellular fractionation protocols.

• Gel selection: Use 10-12% SDS-PAGE gels to achieve optimal separation in the 37-45 kDa range where MRPL38 is observed .

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 60 minutes with methanol-containing transfer buffer is recommended for optimal protein transfer.

• Blocking conditions: 5% non-fat milk in TBST for 1 hour at room temperature shows best results for minimizing background.

• Antibody incubation: Primary antibody dilutions ranging from 1:500-1:2000 for polyclonal antibodies and 1:2000-1:10000 for monoclonal antibodies have shown success . Overnight incubation at 4°C is recommended.

• Positive controls: A549, LNCaP, Jurkat, and K-562 cells have been validated as positive controls for MRPL38 detection . Mouse brain tissue has also shown reliable MRPL38 expression .

• Expected molecular weight: While the calculated molecular weight is 41 kDa, MRPL38 is typically observed between 37-45 kDa depending on post-translational modifications and experimental conditions .

What considerations are important for immunohistochemical detection of MRPL38?

Successful IHC detection of MRPL38 requires attention to several critical parameters:

• Tissue preparation: Both FFPE and frozen sections have been successfully used. For FFPE sections, deparaffinization and rehydration should be followed by antigen retrieval.

• Antigen retrieval: TE buffer pH 9.0 is recommended as the primary method, though citrate buffer pH 6.0 has also proven effective as an alternative . Heat-induced epitope retrieval (HIER) at 95-98°C for 15-20 minutes typically yields optimal results.

• Antibody dilution: For IHC applications, dilutions between 1:50-1:500 are recommended depending on the specific antibody and tissue type . Optimization through dilution series is advised.

• Detection system: Both HRP-polymer and ABC detection systems have demonstrated good results with MRPL38 antibodies. DAB is the most commonly used chromogen.

• Positive control tissues: Human liver tissue and mouse brain tissue have been validated as reliable positive controls for MRPL38 expression .

• Counterstaining: Hematoxylin counterstaining for 1-2 minutes provides optimal nuclear contrast without obscuring specific staining.

• Evaluation: Both cytoplasmic and mitochondrial staining patterns should be assessed, as MRPL38 is predominantly localized to mitochondria .

How can researchers validate the specificity of MRPL38 antibodies in their experimental system?

To ensure antibody specificity and minimize false results, implement the following validation strategies:

• Multiple antibody approach: Use at least two antibodies from different manufacturers targeting different epitopes of MRPL38. Proteintech (15913-1-AP, 68418-1-Ig) and Sigma-Aldrich (HPA023135) antibodies have been extensively validated .

• Knockdown/knockout controls: Implement siRNA knockdown or CRISPR-Cas9 knockout of MRPL38 to verify signal reduction or elimination. Compare with non-targeting controls to confirm specificity.

• Overexpression controls: Transfect cells with MRPL38 expression vectors to verify increased signal intensity. Tagged constructs can be particularly useful for validation.

• Peptide competition assay: Pre-incubate antibody with immunizing peptide (where available) to confirm signal elimination. Some manufacturers offer blocking peptides specifically for this purpose .

• Co-localization studies: Perform dual immunofluorescence with established mitochondrial markers (e.g., TOMM20, COX4) to confirm expected subcellular localization pattern.

• Molecular weight verification: Confirm that the detected band runs at the expected molecular weight range (37-45 kDa) .

• Cross-species reactivity: If using samples from multiple species, verify reactivity using positive control samples from each species of interest .

What is known about MRPL38 expression in cancer and its potential role in metastasis?

MRPL38 has emerged as a potential factor in cancer development and progression, particularly in metastasis:

• Expression patterns: Studies have shown differential expression of MRPL38 in various cancer types. For example, MRPL38 was identified among genes differentially expressed between autoantibody-negative controls and autoantibody-positive arthralgia patients at risk of developing RA .

• Mitochondrial ribosomal proteins in cancer: MRPL38 belongs to a family of mitochondrial ribosomal proteins (MRPs) implicated in cancer progression. MRPL16 has been found to be upregulated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), with significant differences between LUAD subtypes .

• Metastatic potential: While direct evidence for MRPL38's role in metastasis is still emerging, other MRPs like MRPS18B have been shown to enhance cancer cell migration through EMT induction via chemokine signaling (CXCL12-CXCR4) and increased EMT transcription factor (TWIST2) expression .

• Pathway associations: In breast cancer, pathway enrichment analysis has found that related MRPs such as MRPS18B have roles in cancer-associated pathways including PIP3/AKT, estrogen signaling, cell cycle, and circadian rhythm .

• Biomarker potential: In ovarian cancer, MRPS31 (another mitochondrial ribosomal protein) associates with established biomarkers and correlates with poor survival, suggesting MRPs may have value as prognostic indicators . Similar investigations for MRPL38 are warranted.

• Therapeutic implications: The association of MRPs with cancer progression suggests potential therapeutic targeting, though specific interventions targeting MRPL38 require further investigation.

Researchers should consider these findings when designing studies to investigate MRPL38's role in cancer development and progression .

How does MRPL38 factor into respiratory disease research, particularly COPD?

Recent investigations have identified MRPL38 as potentially relevant in chronic obstructive pulmonary disease (COPD):

• Mitochondrial gene networks: MRPL38 has been identified among hub mitochondrial differentially expressed genes (MitoDEGs) in COPD, appearing alongside other mitochondrial ribosomal proteins (MRPL2, MRPL13, MRPL27, etc.) and mitochondrial respiratory chain components (NDUFS2, NDUFS3, etc.) .

• Diagnostic potential: ROC analysis has shown that a set of 32 hub MitoDEGs (including MRPL38) were significantly better at distinguishing between COPD patients and control individuals compared to non-hub MitoDEGs across multiple RNA-seq datasets of COPD lung tissues .

• Causal relationships: Mendelian Randomization (MR) analyses have been employed to assess whether hub MitoDEGs (including MRPL38) are causally associated with COPD using eQTL data. These analyses have identified several genes with potential causal relationships .

• Risk prediction: Nomogram models have suggested that the risk of COPD increases with the downregulation of specific MitoDEGs in pulmonary macrophages, highlighting the importance of mitochondrial function in COPD pathogenesis .

• Methodological considerations: When studying MRPL38 in respiratory disease, researchers should consider cell-type specific expression patterns, particularly in pulmonary macrophages, epithelial cells, and fibroblasts .

These findings suggest MRPL38 and related mitochondrial genes may play important roles in COPD pathogenesis, warranting further investigation into their potential as diagnostic biomarkers or therapeutic targets .

What are the critical considerations for designing knockdown or overexpression experiments targeting MRPL38?

When designing genetic manipulation experiments for MRPL38:

• siRNA/shRNA design: Target conserved regions of MRPL38 mRNA. Use at least 3 independent siRNA sequences to control for off-target effects. Common siRNA transfection methods include lipofection for cell lines and electroporation for primary cells.

• CRISPR-Cas9 strategies: Consider using inducible CRISPR systems for MRPL38 knockout, as complete knockout may affect mitochondrial function and cell viability. Design gRNAs targeting early exons to maximize disruption.

• Rescue experiments: Include rescue conditions with wild-type MRPL38 expression constructs to confirm phenotype specificity. Consider codon-optimized constructs resistant to siRNA targeting.

• Overexpression systems: Use expression vectors with physiologically relevant promoters to avoid artifacts from excessive overexpression. Tagged constructs (e.g., FLAG, HA) can facilitate detection and immunoprecipitation experiments.

• Functional readouts: Assess mitochondrial translation efficiency, respiration rates, and ATP production following MRPL38 manipulation. Consider mitochondrial morphology analysis through confocal microscopy.

• Temporal considerations: Monitor phenotypes at multiple time points, as acute versus chronic alterations in MRPL38 levels may yield different cellular responses.

• Cell-type considerations: The effects of MRPL38 modulation may vary between cell types based on their reliance on mitochondrial function. Include multiple relevant cell types in your experimental design.

How can researchers investigate MRPL38 protein interactions and post-translational modifications?

To characterize MRPL38 protein interactions and modifications:

• Co-immunoprecipitation: Use validated antibodies like 68418-1-Ig (demonstrated effective for IP at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) . Consider crosslinking approaches to capture transient interactions.

• Proximity labeling: BioID or APEX2 fusion with MRPL38 can identify proximal proteins in the mitochondrial ribosome and other potential interaction partners.

• Mass spectrometry: Both targeted and untargeted MS approaches can identify post-translational modifications. Enrichment strategies for phosphorylated, acetylated, or ubiquitinated peptides may be necessary for low-abundance modifications.

• Mitochondrial isolation: For studying MRPL38 in its native context, optimize mitochondrial isolation protocols using differential centrifugation or commercial kits.

• Structural analysis: Consider cryo-EM approaches to understand MRPL38's position and interactions within the mitochondrial ribosome structure.

• Functional interaction studies: Assess how MRPL38 knockdown affects the stability and function of other mitochondrial ribosomal proteins to identify functional dependencies.

• Posttranslational modification site mapping: If modifications are identified, create site-directed mutants (e.g., phosphomimetic or phospho-null) to assess functional consequences.

What are the methodological approaches for studying MRPL38 in animal models of disease?

For investigating MRPL38 in animal disease models:

• Model selection: Based on available data showing reactivity in mouse, rat, and pig samples , these species represent viable models for MRPL38 studies. Consider disease relevance when selecting your model.

• Genetic approaches:

  • Conditional knockout systems (e.g., Cre-loxP) may be preferable to global knockouts if MRPL38 disruption affects development

  • Tissue-specific knockout can help isolate phenotypes relevant to specific diseases

  • CRISPR-mediated knockin of tagged MRPL38 facilitates tracking of the protein in vivo

• Detection methods: For immunohistochemical detection in mouse tissue, validated antibodies with demonstrated mouse reactivity should be used at optimized dilutions (1:50-1:500) . Antigen retrieval with TE buffer pH 9.0, or alternatively with citrate buffer pH 6.0, is recommended.

• Xenograft models: In cancer research, xenograft models have proven valuable for studying MRPs. For example, MRPS18B overexpression in mouse endometrial cancer xenografts produced larger and more vascularized tumors, while prostate cancer zebrafish xenografts with altered MRP expression showed differential tumor growth and migration patterns .

• Disease-specific considerations:

  • For cancer models: assess tumor growth, invasion, metastasis, and angiogenesis

  • For COPD models: evaluate lung function, inflammation, and mitochondrial parameters

  • For neurodegenerative models: examine mitochondrial dysfunction in neuronal tissues

• Multi-omics approaches: Integrate transcriptomics, proteomics, and metabolomics to comprehensively assess the impact of MRPL38 modulation in disease models.

• Translational relevance: Design animal studies with clear translational endpoints that could inform human clinical research or therapeutic development.

What are common issues encountered when working with MRPL38 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with MRPL38 antibodies:

• High background in Western blots:

  • Solution: Increase blocking time (2 hours at room temperature), use 5% BSA instead of milk for blocking, increase washing duration (5 x 5 minutes), and optimize antibody dilution (start with manufacturer's recommendation and adjust as needed) .

• Multiple bands in Western blot:

  • Solution: Verify sample preparation (add fresh protease inhibitors), optimize SDS-PAGE conditions, and consider testing alternative antibodies. Some non-specific bands may represent isoforms or post-translationally modified forms of MRPL38 .

• Weak or no signal in IHC:

  • Solution: Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0), increase antibody concentration, extend incubation time (overnight at 4°C), and use amplification systems like TSA (tyramide signal amplification) .

• Inconsistent cell staining in IF:

  • Solution: Ensure proper fixation (4% paraformaldehyde for 15 minutes), optimize permeabilization (0.1-0.5% Triton X-100), and use confocal microscopy to visualize mitochondrial localization with appropriate Z-stack imaging.

• Poor IP efficiency:

  • Solution: Increase antibody amount (4.0 μg for IP), extend incubation time (overnight at 4°C with rotation), optimize lysis conditions (consider NP-40 or CHAPS-based buffers for mitochondrial proteins), and pre-clear lysates thoroughly .

• Batch-to-batch variability:

  • Solution: Purchase larger antibody lots for long-term projects, validate each new lot against previous lots, and maintain consistent experimental conditions.

• Species cross-reactivity issues:

  • Solution: Verify the antibody's validated species reactivity and consider using species-specific positive controls to confirm reactivity before proceeding with experimental samples.

How can researchers validate results from gene expression studies involving MRPL38?

When conducting and validating gene expression studies involving MRPL38:

• Multi-method validation: Confirm RNA-seq or microarray findings with qRT-PCR using validated primer sets spanning exon-exon junctions. For protein-level validation, use Western blot and IHC with validated antibodies .

• Appropriate controls: Include tissue/cell-specific positive controls where MRPL38 expression is well-established (e.g., A549 cells, LNCaP cells, human liver tissue, mouse brain tissue) .

• Statistical considerations: For differential expression analysis, apply appropriate statistical methods with multiple testing correction. A minimum fold change threshold (often ≥2-fold) combined with statistical significance (p<0.05) provides robust identification of differentially expressed genes .

• Cellular heterogeneity: Consider single-cell approaches when working with heterogeneous tissues to resolve cell type-specific expression patterns. This is particularly relevant for tissues with multiple cell populations (e.g., lung, liver).

• Functional validation: Complement expression data with functional studies using siRNA knockdown or overexpression to establish causality for observed phenotypes.

• Pathway analysis: Place MRPL38 expression changes in biological context through pathway analysis. MRPL38 has been identified in pathways related to mitochondrial translation and has been implicated in cancer-related pathways .

• Cross-species validation: When possible, validate findings across multiple species to strengthen evolutionary conservation arguments for biological importance .

This comprehensive approach ensures robust validation of gene expression findings related to MRPL38.

What emerging technologies might advance our understanding of MRPL38 function and regulation?

Several cutting-edge technologies show promise for elucidating MRPL38 biology:

• Cryo-electron microscopy: High-resolution structural analysis of mitochondrial ribosomes can reveal MRPL38's precise position and interactions within the ribosomal complex, potentially identifying novel interaction partners and functional domains.

• Single-cell multi-omics: Integration of transcriptomics, proteomics, and metabolomics at the single-cell level can reveal cell type-specific roles of MRPL38 in both normal and pathological contexts, particularly important in heterogeneous tissues.

• Spatial transcriptomics/proteomics: These technologies can map MRPL38 expression patterns within tissue microenvironments, potentially revealing regional variations in expression that correlate with disease progression zones.

• CRISPR screening: Genome-wide or targeted CRISPR screens can identify genetic interactions with MRPL38, revealing synthetic lethal relationships and potential therapeutic vulnerabilities in disease contexts.

• Mitoribosome profiling: Adaptation of ribosome profiling techniques specifically for mitochondrial ribosomes can reveal MRPL38's role in translational regulation of specific mitochondrial mRNAs.

• Live-cell imaging: Development of specific tools for visualizing MRPL38 dynamics in living cells (e.g., CRISPR knock-in of fluorescent tags at the endogenous locus) can provide insights into its spatiotemporal regulation.

• Patient-derived organoids: These advanced 3D culture systems can enable the study of MRPL38 in more physiologically relevant models of human disease, bridging the gap between cell lines and in vivo models.

What are the key unanswered questions regarding MRPL38 that researchers should prioritize?

Several critical knowledge gaps remain in our understanding of MRPL38:

• Specific mitochondrial translational targets: Does MRPL38 preferentially affect the translation of specific mitochondrial mRNAs? Ribosome profiling studies comparing wild-type and MRPL38-depleted cells could address this question.

• Post-translational regulation: What post-translational modifications regulate MRPL38 function, and how do these change in disease states? Comprehensive PTM mapping by mass spectrometry would provide valuable insights.

• Non-canonical functions: Does MRPL38 have functions beyond its role in mitochondrial translation? Some mitochondrial ribosomal proteins have been found to have additional roles in processes like apoptosis and cellular stress responses.

• Tissue-specific roles: Does MRPL38 have specialized functions in tissues with high mitochondrial content (e.g., heart, brain, muscle)? Tissue-specific knockout models could help address this question.

• Role in disease pathogenesis: Is MRPL38 dysregulation a cause or consequence of disease processes like cancer progression and COPD? Carefully designed time-course studies and causal inference approaches like Mendelian Randomization can help clarify this relationship .

• Therapeutic targeting: Can MRPL38 be specifically targeted for therapeutic purposes in diseases where its dysregulation contributes to pathology? High-throughput screening for modulators of MRPL38 expression or function may identify potential therapeutic agents.

• Evolutionary considerations: How has MRPL38 function evolved across species, and what can this tell us about its fundamental biological roles? Comparative studies across diverse taxonomic groups could provide evolutionary context for current findings.