MRPS15 Antibody

What is MRPS15 and what are its cellular functions?

MRPS15 (Mitochondrial Ribosomal Protein S15) is a 257 amino acid protein belonging to the ribosomal protein S15P family. Traditionally recognized as a component of the mitochondrial ribosome small subunit, recent research has revealed that MRPS15 also functions in the cytosol, particularly during cellular stress conditions.

MRPS15's dual localization serves important cellular functions:

• In mitochondria: Essential component of the mitochondrial ribosome, necessary for mitochondrial protein synthesis and energy production

• In cytosol: Interacts with cytosolic ribosomes during stress conditions, particularly endoplasmic reticulum (ER) stress

• Specialized function: Acts as an activator of Internal Ribosome Entry Site (IRES)-dependent translation during stress

• Stress response mediator: MRPS15-containing ribosomes specialize in translating mRNAs involved in the unfolded protein response

Mass spectrometry and localization studies have confirmed that while most MRPS15 co-localizes with mitochondria (stained with Mitotracker), a small portion is located in the cytosol, with this cytosolic fraction significantly increasing in tunicamycin-treated cells experiencing ER stress .

What types of MRPS15 antibodies are available and what are their specifications?

Several MRPS15 antibodies are commercially available with different specifications to suit various research applications:

Additionally, antibodies targeting different epitopes are available, including:

• N-terminal region antibodies

• C-terminal region antibodies

• Internal region antibodies (AA 35-84, 51-100, 110-159, etc.)

When selecting an MRPS15 antibody, consider your experimental application, species of interest, and the specific region of MRPS15 you want to target. The observed molecular weight is typically between 25-30 kDa .

How should MRPS15 antibodies be used for different applications?

Optimal use of MRPS15 antibodies varies by application. Here are evidence-based methodological guidelines:

Western Blotting (WB):

• Recommended dilution: 1:500-1:2000

• Expected band size: 25-30 kDa

• Positive control cell lines: HeLa, Raji, U-87 MG, MCF-7

• Positive control tissues: Mouse liver, human breast carcinoma

• Protocol note: Use standard ECL detection methods

Immunohistochemistry (IHC):

• Recommended dilution: 1:20-1:200

• Antigen retrieval: Use citrate buffer pH 6.0 or TE buffer pH 9.0

• Positive control tissues: Human breast carcinoma, human spleen

• Protocol note: Heat-mediated antigen retrieval is essential

Immunofluorescence/Immunocytochemistry (IF/ICC):

• Recommended dilution: 1:200-1:800

• Positive control cells: HeLa, U-87 MG

• Protocol note: For dual localization studies, co-staining with Mitotracker is valuable

Immunoprecipitation (IP):

• Recommended amount: 0.5-4.0 μg antibody for 1.0-3.0 mg total protein lysate

• Positive control sample: Mouse brain tissue

• Protocol note: Include IgG controls to assess non-specific binding

All applications benefit from proper optimization in your specific experimental system .

How can I design experiments to study MRPS15's dual localization in mitochondria and cytosol?

To effectively study MRPS15's dual localization, implement a multi-technique approach:

• Subcellular Fractionation and Western Blotting:

  • Separate cytosolic and mitochondrial fractions using differential centrifugation

  • Analyze MRPS15 levels in each fraction by Western blotting

  • Include fraction-specific markers: mitochondrial (VDAC/COX IV) and cytosolic (GAPDH/β-actin)

  • Compare MRPS15 distribution in stressed vs. unstressed conditions

  • Note: In cardiomyocytes, ER stress (tunicamycin treatment) increases cytosolic MRPS15

• Confocal Microscopy with Co-localization Analysis:

  • Co-stain cells with anti-MRPS15 antibody and Mitotracker

  • Use confocal microscopy to visualize subcellular distribution

  • Perform quantitative co-localization analysis

  • Look for green signal (MRPS15) outside mitochondria in merged panels

  • Compare co-localization coefficients between stressed and unstressed cells

• Proximity Ligation Assay (PLA):

  • Perform PLA using anti-MRPS15 and anti-ribosomal protein (e.g., RPS7) antibodies

  • Quantify interaction dots in the cytosol

  • Compare PLA signal between stressed and unstressed cells

  • Note: Increased cytosolic PLA signal indicates MRPS15-ribosome interaction outside mitochondria

• Co-Immunoprecipitation (Co-IP):

  • Immunoprecipitate MRPS15 from cytosolic fractions (after mitochondria depletion)

  • Probe for co-precipitated ribosomal proteins (RPS2/RPL10A)

  • Include both stressed and unstressed conditions

  • Include proper controls (IgG, input samples)

This multi-faceted approach provides robust evidence for MRPS15's dynamic redistribution between mitochondrial and cytosolic compartments during cellular stress responses.

What controls should be included when validating MRPS15 antibody specificity?

Proper validation of MRPS15 antibody specificity requires comprehensive controls:

• Western Blot Validation:

  • Positive controls: Use cell lines with confirmed MRPS15 expression (HeLa, Raji, U-87 MG)

  • Negative control: MRPS15 knockdown using siRNA (note: complete knockdown may not be achievable due to essential mitochondrial function; ~20% knockdown was maximum achieved in published studies)

  • Size verification: Confirm single band at expected molecular weight (25-30 kDa)

  • Loading controls: Include appropriate housekeeping proteins

  • Multiple antibodies: When possible, confirm findings with antibodies targeting different epitopes

• Immunofluorescence Validation:

  • Secondary antibody-only control: To assess background staining

  • Subcellular marker co-staining: Use Mitotracker to confirm mitochondrial localization

  • Competing peptide control: Pre-incubate antibody with immunizing peptide

  • siRNA knockdown: Compare staining pattern in control vs. knockdown cells

• Immunoprecipitation Validation:

  • IgG control: Use species-matched, non-specific IgG

  • Input control: Include pre-IP sample

  • Reverse IP: Confirm interaction by IP with antibody against interacting protein

  • Specificity control: Compare IP efficiency in control vs. knockdown cells

• Application-Specific Controls:

  • For polysome profiling: Include non-polysomal fractions

  • For IRES activity studies: Include hairpin control vectors

  • For stress response studies: Include both stressed and unstressed conditions

These rigorous validation steps ensure that experimental observations truly reflect MRPS15 biology rather than antibody artifacts.

How can I investigate MRPS15's role in IRES-dependent translation during cellular stress?

To investigate MRPS15's involvement in IRES-dependent translation during stress, implement this comprehensive approach:

• Establish Cellular Stress Model:

  • Induce ER stress in cardiomyocytes (e.g., AC16 cells) using tunicamycin

  • Confirm stress induction by monitoring stress markers (phosphorylated eIF2α, GRP78)

  • Verify global translation inhibition (puromycin incorporation assay)

  • Measure timeline of stress response to determine optimal sampling points

• Bicistronic Reporter Assays:

  • Transduce cells with bicistronic lentivectors containing:

    • First cistron: Renilla luciferase (cap-dependent)

    • Second cistron: Firefly luciferase (IRES-dependent)

    • IRES element of interest between cistrons (e.g., FGF1, IGF1R, VEGFD, EMCV IRESs)

    • Hairpin control vector (negative control)

  • Calculate LucF/LucR ratio to determine IRES activity

  • Compare ratios in stressed vs. unstressed conditions

• Manipulate MRPS15 Levels:

  • Knockdown approach: Transfect siRNA smartpool against MRPS15

    • Monitor knockdown efficiency (expect ~20% maximum)

    • Measure effect on IRES activity using bicistronic reporters

    • Note: Complete knockdown may not be possible due to essential mitochondrial function

  • Overexpression approach: Transduce with lentivector producing cytosolic MRPS15

    • Design construct without mitochondrial targeting sequence (cMRPS15)

    • Confirm overexpression (~25% above endogenous levels)

    • Measure effect on various IRES activities

• RNA Immunoprecipitation and Sequencing:

  • Immunoprecipitate polysomes with anti-MRPS15 antibody

  • Extract and sequence associated mRNAs

  • Compare mRNA profiles between stressed and unstressed conditions

  • Perform pathway analysis on identified mRNAs

  • Note: Research shows MRPS15-associated ribosomes specialize in translating mRNAs involved in unfolded protein response

This approach revealed that MRPS15 functions as an activator of specific IRESs (FGF1 and IGF1R) but not others (VEGFD and EMCV), demonstrating IRES-specific effects that occur primarily during stress conditions .

How can I analyze ribosome heterogeneity with respect to MRPS15 incorporation?

To analyze ribosome heterogeneity with MRPS15 as a focus, apply these advanced methodological approaches:

• Polysome Profiling and Fractionation:

  • Separate polysomal fractions using sucrose gradient ultracentrifugation

  • Collect fractions and analyze by Western blotting for:

    • MRPS15

    • Canonical ribosomal proteins (e.g., RPS7, RPS2, RPL10A)

    • Mitochondrial markers (to exclude mitochondrial contamination)

  • Compare profiles between stressed and unstressed cells

  • Note: Recent research found drastic variation in MRP association with cytosolic polysomes under stress conditions

• Mass Spectrometry Analysis of Ribosome Composition:

  • Isolate polysomes from stressed and unstressed cells

  • Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Quantify relative abundance of all ribosomal and associated proteins

  • Data analysis parameters:

    • Minimum of two unique peptides for identification

    • False discovery rate <1%

    • Compare relative protein abundances between conditions

  • Note: While conventional ribosomal protein composition showed minimal changes, several MRPs (including MRPS15) showed drastic variations in polysomes during stress

• MRPS15-Specific Ribosome Immunoprecipitation:

  • Immunoprecipitate polysomes using anti-MRPS15 antibody

  • Extract associated mRNAs and perform RNA sequencing

  • Compare with total polysomal mRNA population

  • Identify mRNAs preferentially associated with MRPS15-containing ribosomes

  • Perform pathway enrichment analysis on identified mRNAs

  • Results show enrichment for unfolded protein response mRNAs in "MRPS15 ribosomes"

• Ribosome Interaction Visualization:

  • Proximity ligation assay (PLA) between MRPS15 and ribosomal proteins

  • Quantify interaction dots per cell in different conditions

  • Results show increased MRPS15-ribosome interaction during stress

This multi-faceted approach revealed that ribosome heterogeneity through incorporation of typically mitochondrial proteins like MRPS15 into cytosolic ribosomes represents a previously unrecognized mechanism for specialized translation during cellular stress responses .

What insights can be gained from analyzing MRPS15-associated transcripts during stress conditions?

Analysis of MRPS15-associated transcripts during stress conditions reveals crucial insights into specialized translation mechanisms:

• Identification of MRPS15-Specific Translatome:

  • MRPS15 immunoprecipitation followed by RNA sequencing reveals that "MRPS15 ribosomes" preferentially translate a specific subset of mRNAs during ER stress

  • These mRNAs are enriched for transcripts involved in the unfolded protein response (UPR)

  • This demonstrates that MRPS15-containing ribosomes perform specialized translation functions during cellular stress

• IRES-Dependent Translation Regulation:

  • MRPS15 selectively promotes translation through specific IRESs:

    • Significantly activates: FGF1 and IGF1R IRESs

    • Does not affect: VEGFD and EMCV IRESs

  • This IRES selectivity suggests MRPS15 functions through specific molecular interactions rather than as a general IRES activator

  • These effects are observed only in stressed cells, not in unstressed conditions

  • The moderate but significant activation (proportional to the 20-25% knockdown/overexpression achievable) suggests biological relevance

• Stress Response Specialization:

  • During ER stress, global translation is inhibited while IRES-dependent translation is activated

  • MRPS15 participates in this translational reprogramming by:

    • Relocating partially from mitochondria to cytosol

    • Associating with cytosolic ribosomes

    • Promoting translation of stress-response mRNAs

  • This represents a previously unrecognized mitochondria-to-cytosol signaling mechanism

• Pathophysiological Implications:

  • This specialized translation mechanism may be particularly important in tissues prone to ischemic stress, such as cardiac tissue

  • The findings suggest MRPS15 as a potential target for modulating stress responses in cardiovascular disease

  • Cardiac pathologies involving ER stress might involve dysregulation of MRPS15-mediated specialized translation

These insights from MRPS15-associated transcript analysis reveal a sophisticated mechanism of ribosome specialization through incorporation of traditionally mitochondrial components, contributing to our understanding of how cells achieve selective protein synthesis during stress conditions.

How can I address potential discrepancies in MRPS15 detection between different experimental approaches?

When facing discrepancies in MRPS15 detection across different methods, consider these methodological solutions:

• Subcellular Localization Discrepancies:

  • Challenge: Discrepancies between biochemical fractionation and imaging results

  • Solution:

    • Ensure complete mitochondrial depletion in cytosolic fractions

    • Use multiple mitochondrial markers (VDAC, COX IV) to verify fraction purity

    • For imaging, use confocal microscopy with Z-stack analysis

    • Quantify co-localization using software-based analysis (Pearson's coefficient)

    • Remember that only a small portion of MRPS15 (increasing during stress) is cytosolic

• Antibody Performance Variations:

  • Challenge: Different antibodies giving inconsistent results

  • Solution:

    • Optimize antibody dilutions for each application

    • Consider epitope accessibility issues (some epitopes may be masked in certain contexts)

    • Use antibodies targeting different epitopes to validate findings

    • For critical experiments, validate key findings with multiple antibodies

    • Note recommended dilutions and validated applications for each antibody

• MRPS15 Knockdown Efficiency Limitations:

  • Challenge: Limited knockdown efficiency (~20% maximum) due to essential mitochondrial function

  • Solution:

    • Design experiments with realistic expectations of knockdown efficiency

    • Look for proportional effects relative to knockdown achieved

    • Consider inducible or tissue-specific knockdown systems

    • For overexpression, use cytosolic MRPS15 (cMRPS15) without mitochondrial targeting sequence

• Stress Response Timing Discrepancies:

  • Challenge: Variation in timing of MRPS15 redistribution during stress

  • Solution:

    • Perform detailed time-course experiments (0-24h after stress induction)

    • Monitor stress markers (phosphorylated eIF2α, GRP78) alongside MRPS15

    • Consider that peak changes occur at specific time points (4-8h post-treatment in published studies)

    • Cell-type specific differences may exist in response timing

• Quantification Method Discrepancies:

  • Challenge: Different quantification methods yielding variable results

  • Solution:

    • For Western blots, use fluorescence-based detection for better linearity

    • Normalize to appropriate loading controls

    • For microscopy, analyze multiple fields and cells (>50 cells per condition)

    • For mass spectrometry, ensure minimum peptide count (≥2) for reliable quantification

Addressing these potential discrepancies will strengthen the reliability and reproducibility of MRPS15-related findings across different experimental systems.

What are the common challenges in MRPS15 antibody applications and how can they be overcome?

Researchers encounter several challenges when working with MRPS15 antibodies. Here are evidence-based solutions to these common issues:

• Low Signal Intensity in Western Blotting:

  • Challenge: Weak MRPS15 detection despite adequate loading

  • Solutions:

    • Optimize primary antibody concentration (try 1:500 instead of 1:2000)

    • Extend primary antibody incubation (overnight at 4°C)

    • Use enhanced detection systems (high-sensitivity ECL)

    • Increase protein loading (15-30 μg total protein)

    • Try alternative antibodies targeting different epitopes

    • Note that endogenous levels may be low in some cell types

• Background Issues in Immunofluorescence:

  • Challenge: High background obscuring specific MRPS15 signal

  • Solutions:

    • Increase blocking time and concentration (5% BSA, 1-2 hours)

    • Add 0.1-0.3% Triton X-100 for better antibody penetration

    • Reduce primary antibody concentration (try 1:500 instead of 1:200)

    • Include additional washing steps (5x 5 minutes)

    • Use confocal microscopy for better signal-to-noise ratio

    • Co-stain with mitochondrial markers to confirm specific localization

• Inefficient Immunoprecipitation:

  • Challenge: Poor MRPS15 pull-down efficiency

  • Solutions:

    • Optimize antibody amount (try 2-4 μg for 1-3 mg lysate)

    • Extend incubation time (overnight at 4°C with rotation)

    • Try different lysis buffers (RIPA vs. NP-40)

    • Pre-clear lysate with Protein A/G beads

    • Cross-link antibody to beads to prevent antibody contamination in eluate

    • Consider antibodies specifically validated for IP applications

• Inconsistent Results in Stress Response Studies:

  • Challenge: Variable MRPS15 redistribution during stress

  • Solutions:

    • Standardize stress induction protocol (tunicamycin concentration and timing)

    • Confirm stress induction by monitoring established markers

    • Perform detailed time-course experiments

    • Consider cell confluence effects (70-80% optimal)

    • Control for cell passage number (use cells within similar passage range)

    • Expect subtle changes rather than dramatic redistribution

• Cross-Reactivity Concerns:

  • Challenge: Potential cross-reactivity with related proteins

  • Solutions:

    • Validate with MRPS15 knockdown controls

    • Compare multiple antibodies targeting different epitopes

    • Perform peptide competition assays

    • Consider MS/MS verification of immunoprecipitated proteins

    • When using antibodies in new species, perform thorough validation

By implementing these specific solutions, researchers can overcome common challenges associated with MRPS15 antibody applications and obtain more reliable, reproducible results in their investigations.

What emerging applications of MRPS15 antibodies show promise for advancing cellular stress research?

Several innovative applications of MRPS15 antibodies show particular promise for advancing our understanding of cellular stress responses:

• Single-Cell Analysis of MRPS15 Dynamics:

  • Combine MRPS15 immunofluorescence with single-cell transcriptomics

  • Map heterogeneous stress responses within cell populations

  • Correlate MRPS15 relocalization with single-cell proteomics

  • This approach could reveal how individual cells within a population differ in their translational stress responses

• MRPS15 Proximity Labeling for Interaction Networks:

  • Develop MRPS15-BioID or APEX2 fusion proteins

  • Map complete interaction networks in unstressed vs. stressed conditions

  • Identify stress-specific MRPS15 partners beyond ribosomal proteins

  • This would expand on current co-IP findings to provide a comprehensive interaction map

• Dynamic MRPS15 Imaging in Living Cells:

  • Create fluorescent protein-tagged MRPS15 constructs

  • Perform live-cell imaging during stress induction

  • Combine with ribosome labeling techniques

  • This approach would provide temporal resolution of MRPS15 redistribution during stress

• Tissue-Specific Analysis in Disease Models:

  • Apply MRPS15 antibodies to tissue sections from ischemic disease models

  • Compare MRPS15 distribution in affected vs. unaffected areas

  • Correlate with markers of stress and cell survival

  • This could reveal the pathophysiological relevance of MRPS15 in disease states

• Therapeutic Target Validation:

  • Use MRPS15 antibodies to monitor effects of compounds modulating stress responses

  • Develop assays for high-throughput screening of stress pathway modulators

  • Validate MRPS15 as a biomarker for cellular stress in pathological samples

  • This application could bridge fundamental research and therapeutic development

These emerging applications leverage MRPS15 antibodies as tools for exploring previously unrecognized aspects of translational control during cellular stress, potentially opening new avenues for understanding and treating stress-related pathologies.

How might integrating MRPS15 antibody techniques with other methodologies enhance our understanding of ribosome heterogeneity?

Integrating MRPS15 antibody techniques with complementary methodologies promises to significantly advance our understanding of ribosome heterogeneity:

• Combination with Cryo-EM Structural Analysis:

  • Immunopurify MRPS15-containing cytosolic ribosomes for cryo-EM

  • Determine structural changes induced by MRPS15 incorporation

  • Map MRPS15 binding sites on the ribosome at near-atomic resolution

  • This would reveal how MRPS15 incorporation might alter ribosome conformation and function

• Integration with Ribosome Profiling:

  • Perform MRPS15 immunoprecipitation followed by ribosome profiling

  • Generate codon-resolution maps of MRPS15-associated translation

  • Compare with conventional ribosome profiling data

  • This approach would reveal how MRPS15-containing ribosomes differ in elongation dynamics and pause sites

• Coupling with Translational Efficiency Measurements:

  • Combine polysome profiling with MRPS15 immunoprecipitation

  • Correlate mRNA association with MRPS15-ribosomes and protein output

  • Measure translation rates of specific mRNAs in the presence/absence of MRPS15

  • This would determine whether MRPS15 affects both mRNA selection and translation rate

• Multi-omics Integration Approaches:

  • Create integrated datasets combining:

    • MRPS15 immunoprecipitation-RNA-seq data

    • Proteomics of stress responses

    • Ribosome composition analysis

    • mRNA structural analyses (particularly of IRES elements)

  • Apply machine learning to identify patterns and predictive features

  • This computational approach could reveal rules governing MRPS15-mediated translation

• Combining with RNA Structure Probing:

  • Apply SHAPE or DMS-seq to mRNAs associated with MRPS15-containing ribosomes

  • Identify structural features recognized by MRPS15-ribosomes

  • Focus on IRES elements with differential sensitivity to MRPS15

  • This would reveal mechanistic details of how MRPS15 promotes IRES-dependent translation

These integrated approaches would transform our understanding of ribosome heterogeneity from a descriptive observation to a mechanistically understood process, potentially revealing new principles of translational control during cellular stress responses.