MRPL15 Antibody

What is MRPL15 and why is it significant in cancer research?

MRPL15 (Mitochondrial Ribosomal Protein L15) is a 39S subunit protein that belongs to the EcoL15 ribosomal protein family. It is encoded by nuclear genes and plays a crucial role in protein synthesis within the mitochondrion. Mitochondrial ribosomes (mitoribosomes) consist of a small 28S subunit and a large 39S subunit, with an estimated 75% protein to rRNA composition compared to prokaryotic ribosomes, where this ratio is reversed .

Proper validation of MRPL15 antibodies is critical for ensuring experimental reliability. A comprehensive validation approach should include:

• Western Blot Analysis: Verify a single band at the expected molecular weight (33-35 kDa for MRPL15) . Test multiple cell lines known to express MRPL15, such as HEK-293, HeLa, HepG2, and MCF-7 cells .

• Positive and Negative Controls: Include positive controls (tissues/cells known to express MRPL15) and negative controls (antibody diluent only) . For instance, rectal tissue has been used as a positive control for MRPL15 expression in immunohistochemistry .

• Knockdown/Knockout Validation: Use siRNA or CRISPR-based approaches to reduce or eliminate MRPL15 expression, then confirm antibody specificity through reduced or absent signal.

• Overexpression Systems: Test antibody specificity in systems overexpressing tagged MRPL15 protein.

• Cross-Reactivity Assessment: Evaluate potential cross-reactivity with related mitochondrial ribosomal proteins, particularly those in the same family.

When publishing results, researchers should clearly document validation methods to enhance reproducibility and confidence in findings .

What are the recommended experimental conditions for Western blotting with MRPL15 antibodies?

Optimal Western blotting conditions for MRPL15 antibodies vary by manufacturer, but general guidelines include:

Sample Preparation:

• Extract proteins from cells using standard lysis buffers containing protease inhibitors

• Load 20-25 μg of total protein per lane

• Include positive control lysates (e.g., HEK-293, HeLa, HepG2, or MCF-7 cells)

Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes using standard protocols

Antibody Incubation:

• Blocking: 5% non-fat milk or BSA in TBST, 1 hour at room temperature

• Primary antibody dilution: Typically 1:2000-1:16000 for rabbit polyclonal antibodies

• Incubation: Overnight at 4°C

• Secondary antibody: HRP-conjugated anti-rabbit IgG (typically 1:5000-1:10000)

Detection:

• Use enhanced chemiluminescence (ECL) detection system

• Expected molecular weight: 33-35 kDa

For troubleshooting, consider optimizing antibody concentration, incubation time, and blocking conditions if nonspecific binding occurs. Some antibodies may require sample-dependent optimization to achieve optimal results .

How can MRPL15 antibodies be utilized to investigate its prognostic value in different cancer types?

MRPL15 antibodies have proven valuable for investigating the protein's prognostic significance across multiple cancer types. To effectively study MRPL15's prognostic value:

• Immunohistochemistry (IHC) Analysis of Tissue Microarrays:

  • Use polyclonal antibodies at optimized dilutions (e.g., 1:150 as reported for ovarian cancer studies)

  • Implement standardized scoring systems based on staining intensity and percentage of positive cells

  • Correlate MRPL15 expression with clinical outcomes using Kaplan-Meier survival analysis

• Multi-cohort Validation Approach:

  • Analyze MRPL15 expression across multiple patient cohorts

  • In NSCLC studies, researchers utilized 8 GEO series (GSE8569, GSE101929, GSE33532, GSE27262, GSE21933, GSE19804, GSE19188, GSE18842) to confirm MRPL15 upregulation

  • For ovarian cancer, GSE51088 and GSE13876 datasets provided validation

• Correlation with Clinicopathological Parameters:

  • Analyze MRPL15 expression in relation to tumor stage, grade, metastasis status

  • In NSCLC, MRPL15 expression correlates with gender, clinical stage, lymph node status, and TP53 mutation status

  • In ovarian cancer, MRPL15 expression relates to HE4 (human epididymis protein 4) levels

• Multivariate Analysis:

  • Use Cox regression models to determine if MRPL15 is an independent prognostic factor

  • Control for confounding variables such as age, stage, and molecular subtypes

Research demonstrates that high MRPL15 expression correlates with poor OS, PFS, and DFS in NSCLC patients (HR = 1.35, log rank P = 3.30E−06) . Similarly, elevated MRPL15 expression associates with unfavorable outcomes in ovarian cancer patients .

What methodological approaches help resolve contradictory findings about MRPL15 expression patterns?

Contradictory findings regarding MRPL15 expression can arise from methodological differences. To address these inconsistencies:

• Standardized Antibody Validation:

  • Implement rigorous antibody validation protocols as described in section 1.3

  • Document antibody catalog numbers, dilutions, and validation methods

  • Consider using multiple antibodies targeting different epitopes of MRPL15

• Multi-omics Integration:

  • Correlate protein expression data (immunoblotting/IHC) with transcriptomic data

  • Analyze MRPL15 expression in relation to copy number variations and methylation status

  • Studies have shown that high MRPL15 expression in ovarian cancer may be associated with gene amplification and hypomethylation

• Context-Specific Analysis:

  • Stratify analyses by cancer subtypes, molecular classifications, or immune profiles

  • MRPL15 expression varies across immune subtypes, showing lowest expression in C3 ovarian cancer

  • In NSCLC, MRPL15 upregulation was notably correlated with smoking history, affecting survival outcomes

• Technical Normalization:

  • Use appropriate housekeeping genes or loading controls

  • Apply batch effect correction in multi-center studies

  • Employ quantitative image analysis for IHC interpretation

• Meta-analysis Approach:

  • Synthesize findings across multiple studies using formal meta-analysis techniques

  • Account for methodological heterogeneity among studies

  • Consider publication bias in interpretation

By implementing these approaches, researchers can better reconcile discrepant findings and develop a more coherent understanding of MRPL15's role in different cancer contexts.

How does MRPL15 contribute to cancer progression and what pathways are implicated?

MRPL15 appears to influence cancer progression through multiple molecular mechanisms, which can be investigated using antibody-based techniques in combination with other methods:

• Metabolism-Related Pathways:

  • KEGG pathway analysis in NSCLC revealed that MRPL15 co-expressed genes participate in oxidative phosphorylation, carbon metabolism, and pyrimidine metabolism

  • Gene set enrichment analysis (GSEA) in ovarian cancer indicated MRPL15 involvement in mTOR signaling

• Cell Cycle and DNA Replication:

  • MRPL15 co-expressed genes are enriched in DNA replication and cell cycle pathways in NSCLC

  • Functional studies suggest its role in promoting cellular proliferation

• Immune Regulation:

  • MRPL15 expression negatively correlates with immune infiltration in NSCLC, including immune scores, stromal scores, and tumor-infiltrating lymphocytes (TILs)

  • In ovarian cancer, MRPL15 shows correlations with CD8+ T cell and dendritic cell proliferation, and with expression of immune regulatory factors like TGFβR1 and IDO1

• Signaling Networks:

  • Functional network analysis indicates that MRPL15 participates in pathways involving several kinases, miRNAs, and transcription factors in NSCLC

  • Co-expression analysis can reveal additional signaling partners

To investigate these mechanisms, researchers can employ:

• Co-immunoprecipitation with MRPL15 antibodies to identify protein interaction partners

• Chromatin immunoprecipitation (ChIP) to study transcriptional regulation

• Immunofluorescence co-localization studies to examine subcellular distribution

• Phospho-specific antibodies to assess activation of downstream signaling pathways

Understanding these mechanisms can inform therapeutic strategies targeting MRPL15 or its associated pathways.

What are the considerations for using MRPL15 antibodies in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence (mIF) allows simultaneous detection of multiple proteins in a single tissue section, providing valuable spatial context for MRPL15 expression. Key considerations include:

• Antibody Selection and Validation:

  • Choose MRPL15 antibodies validated for immunofluorescence applications

  • Verify specificity through appropriate controls

  • Test for potential cross-reactivity with other mitochondrial proteins

• Panel Design:

  • Include markers of mitochondrial function (e.g., TOMM20, COX4)

  • Add cancer-specific markers based on tissue type

  • Consider immune cell markers to study MRPL15's relationship with immune infiltration

  • For NSCLC or ovarian cancer studies, include clinically relevant markers (e.g., HE4 for ovarian cancer)

• Technical Optimization:

  • Determine optimal antibody concentration and incubation conditions

  • Test antigen retrieval methods compatible with all antibodies in the panel

  • Establish appropriate blocking procedures to minimize background

  • Validate signal specificity in multiplex context

• Signal Assignment and Spectral Unmixing:

  • Use appropriate fluorophores with minimal spectral overlap

  • Implement controls for autofluorescence, especially in tissues with high autofluorescence (e.g., lung)

  • Apply spectral unmixing algorithms if needed

• Quantitative Analysis:

  • Develop algorithms to quantify MRPL15 expression at subcellular resolution

  • Analyze co-localization with other markers of interest

  • Implement spatial analysis to study MRPL15 expression in relation to tumor microenvironment features

Multiplexed approaches can reveal important insights about MRPL15's spatial relationship with immune cells, which is particularly relevant given the protein's negative correlation with immune infiltration in cancer .

How can researchers design experiments to investigate MRPL15's role in therapeutic resistance?

To investigate MRPL15's potential role in therapeutic resistance, researchers can design experiments using MRPL15 antibodies in the following approaches:

• Expression Analysis in Treatment-Resistant Models:

  • Compare MRPL15 expression before and after development of resistance using Western blot or IHC

  • Analyze paired pre-treatment and post-relapse patient samples

  • Correlate MRPL15 expression with response to specific therapies

• MRPL15 Modulation Studies:

  • Create MRPL15 knockdown and overexpression models in cancer cell lines

  • Assess sensitivity to chemotherapy, targeted therapy, or immunotherapy

  • Monitor changes in mitochondrial function, metabolism, and cell survival pathways

• Pathway Analysis:

  • Investigate how MRPL15 expression affects known resistance mechanisms:

    • Metabolic reprogramming (assess markers like PKM2, LDHA)

    • DNA repair (examine γH2AX, RAD51, BRCA1/2)

    • Apoptotic signaling (measure BCL2, BAX, cleaved caspases)

  • Use phospho-specific antibodies to assess activation of survival pathways (AKT, ERK, etc.)

• In vivo Models:

  • Develop patient-derived xenografts with varying MRPL15 expression levels

  • Monitor response to therapy and correlate with MRPL15 status

  • Use IHC to assess MRPL15 expression in different tumor regions

• High-Throughput Approaches:

  • Implement antibody-based reverse-phase protein arrays (RPPA) to analyze signaling networks

  • Use CRISPR screens combined with MRPL15 modulation to identify synthetic lethal interactions

Research in NSCLC has shown that MRPL15 participates in metabolism-related pathways, including oxidative phosphorylation and pyrimidine metabolism , which are often altered in resistant tumors. Additionally, its correlation with poor survival in cancer patients suggests a potential role in aggressive, treatment-resistant disease .

What are the critical factors for optimizing immunohistochemistry protocols with MRPL15 antibodies?

Optimizing immunohistochemistry (IHC) protocols for MRPL15 antibodies requires attention to several critical factors:

• Tissue Fixation and Processing:

  • Optimal fixation: 10% neutral buffered formalin for 24-48 hours

  • Consistent processing parameters to ensure reproducibility

  • Paraffin embedding and sectioning at 5μm thickness

• Antigen Retrieval:

  • Test multiple methods (heat-induced epitope retrieval in citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Optimize duration and temperature of antigen retrieval

  • Document optimal conditions for each antibody

• Antibody Selection and Dilution:

  • For polyclonal antibodies, dilution ranges of 1:100-1:200 are common starting points

  • In ovarian cancer studies, a 1:150 dilution of polyclonal MRPL15 antibody from Atlas Antibodies was effective

  • Perform titration experiments to determine optimal concentration

• Detection System:

  • Consider signal amplification systems for low-abundance proteins

  • The streptavidin-peroxidase method has been successfully used for MRPL15 detection

  • Use DAB (3,3'-diaminobenzidine) as chromogen for consistent results

• Scoring and Interpretation:

  • Implement a standardized scoring system

  • In published studies, MRPL15 staining has been scored on a scale of 0-3 (0: no stain, 1: light-yellow, 2: brown-yellow, 3: dark-brown)

  • Calculate composite scores incorporating both intensity and percentage of positive cells

  • Ensure blinded assessment by multiple observers

• Controls:

  • Include positive control tissues (rectal tissue has been used for MRPL15)

  • Use negative controls (primary antibody omitted)

  • Consider on-slide positive and negative controls

Cytoplasmic staining with brown-yellow particles typically indicates MRPL15 positivity, consistent with its mitochondrial localization .

How can researchers accurately quantify MRPL15 protein levels in complex biological samples?

Accurate quantification of MRPL15 in complex samples requires rigorous methodology:

• Western Blot Quantification:

  • Use serial dilutions of recombinant MRPL15 to create a standard curve

  • Apply internal loading controls (β-actin, GAPDH, or mitochondrial proteins like VDAC1)

  • Implement band density analysis with linear dynamic range validation

  • Consider normalization to total protein (Ponceau S or REVERT staining)

• Mass Spectrometry-Based Approaches:

  • Implement targeted proteomics (SRM/MRM) for absolute quantification

  • Use stable isotope-labeled peptide standards specific to MRPL15

  • Monitor multiple unique peptides derived from MRPL15

  • Apply appropriate data normalization strategies

• ELISA/Immunoassays:

  • Develop sandwich ELISA using two antibodies recognizing different MRPL15 epitopes

  • Validate assay sensitivity, specificity, and dynamic range

  • Include matrix-matched calibration standards

• Capillary Western Immunoassay (Wes):

  • Provides higher sensitivity and reproducibility than traditional Western blot

  • Requires less sample input

  • Allows for greater quantification precision

• Image-Based Quantification:

  • For IHC/IF, use digital image analysis with validated algorithms

  • Implement machine learning approaches for tissue segmentation

  • Calculate H-scores or other semi-quantitative metrics

When developing quantification strategies, consider:

• Sample preparation consistency

• Potential interfering substances

• Batch effects and inter-assay variability

• Lower limit of quantification and upper limit of linearity

Quantitative data should be reported with appropriate statistical analyses and measures of technical and biological variation.

What experimental controls are essential when studying MRPL15 in different cellular compartments?

Given MRPL15's mitochondrial localization, proper controls are essential when studying its distribution across cellular compartments:

• Subcellular Fractionation Controls:

  • Verify fractionation purity using compartment-specific markers:

    • Mitochondria: TOMM20, COX4, or VDAC

    • Cytosol: GAPDH or tubulin

    • Nucleus: Lamin B1 or Histone H3

    • Endoplasmic reticulum: Calnexin or PDI

  • Include samples treated with respiratory chain inhibitors (e.g., rotenone, antimycin A) to assess mitochondrial integrity

• Immunofluorescence Microscopy Controls:

  • Co-staining with established mitochondrial markers (MitoTracker, TOMM20)

  • Include additional organelle markers to assess potential non-canonical localization

  • Use super-resolution microscopy to precisely map MRPL15 within mitochondria

  • Implement Z-stack imaging to capture the three-dimensional distribution

• Genetic Controls:

  • MRPL15 knockdown/knockout cells as negative controls

  • MRPL15-GFP fusion constructs with mitochondrial targeting sequence validation

  • Point mutants affecting mitochondrial localization signals

• Physiological Perturbation Controls:

  • Mitochondrial stress inducers (CCCP, oligomycin)

  • Hypoxia/normoxia comparisons

  • Metabolic substrate alterations

• Antibody Validation for Localization Studies:

  • Multiple antibodies targeting different epitopes

  • Peptide competition assays

  • Signal specificity in fractionated samples

These controls are particularly important when investigating whether MRPL15 might have non-canonical functions outside the mitochondria, which could be relevant to its role in cancer progression beyond mitochondrial translation.

How can MRPL15 antibodies be integrated into immuno-oncology research frameworks?

Integrating MRPL15 antibodies into immuno-oncology research can provide insights into the relationship between mitochondrial function and anti-tumor immunity:

• Tumor Microenvironment Analysis:

  • Co-stain tumor sections for MRPL15 and immune cell markers

  • Assess spatial relationships between MRPL15-high tumor regions and immune infiltrates

  • Studies show MRPL15 expression negatively correlates with immune scores, stromal scores, and TILs in NSCLC

• Immune Checkpoint Correlation Studies:

  • Analyze relationship between MRPL15 expression and immune checkpoint molecules (PD-L1, CTLA-4)

  • In ovarian cancer, MRPL15 correlates with expression of immune regulatory factors like TGFβR1 and IDO1

• T-cell Function Assays:

  • Compare T-cell metabolic profiles when co-cultured with MRPL15-high versus MRPL15-low tumor cells

  • Assess impact on T-cell activation, proliferation, and effector functions

• Cancer Immune Subtyping:

  • Use MRPL15 as part of a panel to classify tumors by metabolic-immune phenotype

  • MRPL15 shows differential expression across immune subtypes, with lowest expression in C3 ovarian cancer

• Therapeutic Response Prediction:

  • Correlate MRPL15 expression with response to immunotherapies

  • Develop combinatorial biomarker approaches incorporating MRPL15 status

Research design should employ:

• Multiplex immunofluorescence to preserve spatial context

• Single-cell approaches to resolve heterogeneity

• Functional assays to establish causality beyond correlation

The negative correlation between MRPL15 and immune infiltration suggests it may contribute to an immunosuppressive microenvironment, potentially through metabolic reprogramming mechanisms .

What are the methodological approaches for investigating MRPL15's role in mitochondrial dysfunction in cancer?

To investigate MRPL15's role in mitochondrial dysfunction and its implications for cancer:

• Mitochondrial Protein Synthesis Assessment:

  • Pulse-chase labeling with 35S-methionine in the presence of cytoplasmic translation inhibitors

  • Western blot analysis of mitochondrially-encoded proteins

  • Polysome profiling of mitochondrial ribosomes using MRPL15 antibodies

• Mitochondrial Function Analysis:

  • Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Assess mitochondrial membrane potential using potentiometric dyes

  • Quantify ATP production from oxidative phosphorylation

• Mitochondrial Dynamics:

  • Co-immunostaining for MRPL15 and markers of mitochondrial fusion/fission

  • Live-cell imaging to track mitochondrial morphology

  • Electron microscopy to evaluate ultrastructural changes

• ROS Production and Oxidative Stress:

  • Measure mitochondrial ROS using specific indicators

  • Assess oxidative damage markers (protein carbonylation, lipid peroxidation)

  • Analyze antioxidant response pathways

• Mitochondrial-Nuclear Signaling:

  • Investigate retrograde signaling pathways activated by MRPL15 modulation

  • Analyze nuclear gene expression changes using RNA-seq

  • KEGG pathway analysis in NSCLC showed MRPL15 co-expressed genes participate in metabolism-related pathways

• In vivo Metabolic Imaging:

  • PET imaging with metabolism-specific tracers

  • Correlation of imaging data with MRPL15 expression in tumor biopsies

These approaches can help determine whether MRPL15 upregulation in cancer represents a compensatory response to mitochondrial dysfunction or a driver of metabolic reprogramming that promotes tumor growth.

How should researchers design experiments to clarify contradictory findings about MRPL15's interactions with other proteins?

Resolving contradictory findings about MRPL15's protein interactions requires systematic experimental design:

• Optimized Co-Immunoprecipitation (Co-IP) Protocols:

  • Use multiple antibodies targeting different MRPL15 epitopes

  • Test various lysis conditions to preserve native interactions

  • Include appropriate controls (IgG control, input samples)

  • Validate using reverse Co-IP (pull-down with antibody against interacting partner)

• Proximity Ligation Assay (PLA):

  • Visualize protein-protein interactions in situ

  • Provides spatial context for interactions

  • Quantify interaction frequency in different subcellular compartments

• Cross-linking Mass Spectrometry:

  • Apply protein cross-linking prior to immunoprecipitation

  • Use mass spectrometry to identify interaction partners

  • In situ peroxidase labeling followed by mass spectrometry has been used to identify MRPL15 interactors

• Mammalian Two-Hybrid or Split-Luciferase Assays:

  • Test specific binary interactions in live cells

  • Provides quantitative measure of interaction strength

  • Can evaluate effects of mutations or treatments

• FRET/BRET Analysis:

  • Monitor real-time protein interactions in live cells

  • Assess spatial and temporal dynamics of interactions

  • Test physiological stimuli that may regulate interactions

• Validation in Multiple Cell Types:

  • Test interactions in different cellular contexts

  • Compare cancer versus normal cells

  • Include cell lines commonly used in MRPL15 research (HEK-293, HeLa, HepG2, MCF-7)

• Functional Validation:

  • Perform domain mapping to identify interaction regions

  • Create interaction-deficient mutants

  • Assess functional consequences of disrupting specific interactions

These methodological approaches can help reconcile contradictory findings by identifying context-dependent interactions and technical factors that may influence experimental outcomes.

How can single-cell approaches advance our understanding of MRPL15 heterogeneity in tumor tissues?

Single-cell technologies offer unprecedented opportunities to explore MRPL15 heterogeneity:

• Single-Cell Proteomics Applications:

  • Single-cell mass cytometry (CyTOF) incorporating MRPL15 antibodies

  • Imaging mass cytometry for spatial assessment of MRPL15 heterogeneity

  • Microfluidic single-cell Western blotting

• Spatial Transcriptomics Integration:

  • Correlate MRPL15 protein expression with transcriptomic profiles

  • Implement combined protein-RNA detection methods (CITE-seq)

  • Develop computational methods to integrate protein and RNA data

• Lineage Tracing with MRPL15 Profiling:

  • Track clonal evolution and MRPL15 expression changes

  • Identify evolutionary trajectories of MRPL15-high clones

  • Assess competitive fitness of cells with varying MRPL15 levels

• Single-Cell Functional Assays:

  • Correlate MRPL15 levels with metabolic profiles at single-cell resolution

  • Implement microfluidic approaches to measure mitochondrial function

  • Link MRPL15 expression to drug sensitivity in individual cells

• Artificial Intelligence-Assisted Image Analysis:

  • Develop deep learning algorithms for single-cell segmentation in MRPL15-stained tissues

  • Extract multidimensional features from single-cell MRPL15 expression patterns

  • Identify rare cell populations with unique MRPL15 characteristics

These approaches can address key questions about MRPL15 in cancer:

• Does MRPL15 expression mark specific tumor subpopulations with distinct functional properties?

• How does MRPL15 heterogeneity relate to therapeutic resistance?

• Are there rare MRPL15-expressing cells that drive tumor progression?

The integration of single-cell MRPL15 profiling with functional characterization can provide crucial insights into its role in tumor heterogeneity and progression.

What methodological considerations apply when evaluating MRPL15 as a potential therapeutic target?

Evaluating MRPL15 as a therapeutic target requires systematic assessment:

• Target Validation Approaches:

  • Genetic manipulation studies (CRISPR knockout, shRNA knockdown)

  • Rescue experiments to confirm specificity

  • Patient-derived xenograft models with MRPL15 modulation

• Differential Dependency Assessment:

  • Compare effects of MRPL15 inhibition in cancer versus normal cells

  • Evaluate synthetic lethality contexts

  • Identify cancer subtypes most dependent on MRPL15 function

• Structural Biology Integration:

  • Determine MRPL15 structure and functional domains

  • Identify potential binding pockets for small molecule development

  • Structure-based drug design approaches

• Combinatorial Therapeutic Approaches:

  • Test MRPL15 inhibition in combination with standard therapies

  • Evaluate synergy with metabolic inhibitors

  • Explore combinations with immunotherapies, given MRPL15's negative correlation with immune infiltration

• Biomarker Development:

  • Identify patient populations likely to benefit from MRPL15-targeted therapies

  • Develop companion diagnostic approaches

  • Establish predictive biomarkers of response

• Pharmacodynamic Assay Development:

  • Create assays to measure on-target engagement

  • Develop methods to monitor mitochondrial translation efficiency

  • Implement tissue-based biomarkers for clinical trials

Research in ovarian cancer has identified MRPL15 as a potential therapeutic target due to its association with poor prognosis and its role in cellular proliferation pathways . Similarly, its upregulation in NSCLC and correlation with unfavorable outcomes suggest therapeutic potential in lung cancer .

Carefully designed experimental approaches can help determine whether MRPL15 represents a viable cancer therapeutic target with an acceptable therapeutic window.