MRPS2 Antibody

What is MRPS2 and what is its biological significance?

MRPS2 (Mitochondrial Ribosomal Protein S2) is a nuclear-encoded protein that forms part of the small 28S subunit of mitochondrial ribosomes. It belongs to the universal ribosomal protein uS2 family and plays a critical role in mitoribosome formation, stability, and mitochondrial translation . Mammalian mitoribosomes differ significantly from prokaryotic ribosomes, with an estimated 75% protein to rRNA composition (compared to prokaryotic ribosomes where this ratio is reversed) . The calculated and observed molecular weight of human MRPS2 is approximately 33kDa .

Functionally, MRPS2 is essential for proper mitochondrial protein synthesis, which impacts cellular energy production through oxidative phosphorylation. Research targeting this protein helps elucidate mechanisms of mitochondrial translation and associated pathologies.

What are the common applications for MRPS2 antibodies in research?

MRPS2 antibodies are versatile research tools employed across multiple experimental techniques:

These applications allow researchers to investigate MRPS2 expression levels, cellular/subcellular localization, and interactions with other proteins across different experimental contexts .

How do I select the appropriate MRPS2 antibody for my experiments?

Selection of an appropriate MRPS2 antibody should be based on several critical factors:

• Epitope specificity: Consider whether you need an antibody targeting the N-terminal, C-terminal, or full-length (AA 1-296) protein. Different epitopes may expose different functional domains or be more accessible depending on your experimental conditions .

• Host species: Rabbit-derived polyclonal antibodies are most common for MRPS2 detection, but mouse-derived antibodies are also available. Select based on compatibility with your secondary detection system and to avoid cross-reactivity with other antibodies in multi-labeling experiments .

• Species reactivity: Verify that the antibody recognizes MRPS2 in your species of interest. Most commercial antibodies target human MRPS2, though some cross-react with mouse or rat homologs .

• Application compatibility: Ensure the antibody has been validated for your specific application (WB, ELISA, IHC, etc.). Some antibodies perform well in denatured conditions (WB) but poorly in native conditions (IHC) .

• Conjugation: Determine whether you need an unconjugated antibody or one directly labeled with fluorophores (FITC), enzymes (HRP), or tags (Biotin) based on your detection method .

What is the optimal protocol for detecting MRPS2 using Western blotting?

For optimal MRPS2 detection using Western blotting, follow these methodological recommendations:

• Sample preparation:

  • Extract total protein from cells/tissues using a buffer containing protease inhibitors

  • Include mitochondrial enrichment steps for enhanced detection

  • Denature samples in loading buffer containing SDS and reducing agent at 95°C for 5 minutes

• Gel electrophoresis:

  • Use 12-15% SDS-PAGE gels (15% recommended for optimal resolution near 33kDa)

  • Load 20-50μg of total protein per lane

  • Include molecular weight markers spanning 20-50kDa range

• Transfer and blocking:

  • Transfer to PVDF membranes (0.2μm pore size) at 100V for 1 hour

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation:

  • Dilute primary MRPS2 antibody at 1:1000-1:5000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST (5 minutes each)

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Wash 3-5 times with TBST

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Expect a band at approximately 33kDa

  • Include positive controls (tissue/cells known to express MRPS2)

This protocol can be further optimized based on specific antibody recommendations from manufacturers.

How can I validate the specificity of my MRPS2 antibody?

Rigorous validation of MRPS2 antibody specificity is crucial for generating reliable research data. Employ the following comprehensive validation strategy:

• Positive and negative controls:

  • Use tissues/cells with confirmed high MRPS2 expression as positive controls

  • Include tissues/cells with low or no MRPS2 expression as negative controls

  • Consider using MRPS2 knockout or knockdown samples as definitive negative controls

• Molecular weight verification:

  • Confirm single band at expected molecular weight (33kDa)

  • Investigate any additional bands for potential splice variants or cross-reactivity

• Peptide competition assay:

  • Pre-incubate antibody with excess purified MRPS2 recombinant protein

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific signal should be abolished in the antibody+peptide sample

• Orthogonal method comparison:

  • Compare results with alternative antibodies targeting different MRPS2 epitopes

  • Correlate protein detection with mRNA expression data

  • Use mass spectrometry to confirm identity of immunoprecipitated protein

• Signal correlation with biological context:

  • Verify appropriate subcellular localization (mitochondrial)

  • Confirm expected expression patterns across tissues/cell types

  • Validate signal modulation under conditions known to affect MRPS2 expression

This multi-faceted approach provides robust evidence of antibody specificity and experimental reliability.

What are effective strategies for optimizing MRPS2 immunostaining in fixed tissues?

Successful MRPS2 immunostaining in fixed tissues requires careful optimization of several parameters:

• Fixation method selection:

  • For formalin-fixed paraffin-embedded (FFPE) tissues: Use 10% neutral-buffered formalin, fix for 24-48 hours

  • For frozen sections: Fix with 4% paraformaldehyde for 10-15 minutes

  • Consider dual fixation with glutaraldehyde for enhanced mitochondrial preservation

• Antigen retrieval optimization:

  • Test heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0) at 95-100°C for 20 minutes

    • EDTA buffer (pH 8.0) at 95-100°C for 20 minutes

    • Tris-EDTA buffer (pH 9.0) at 95-100°C for 20 minutes

  • Enzymatic retrieval may be less effective for mitochondrial proteins

• Blocking and permeabilization:

  • Block with 5-10% normal serum from secondary antibody host species

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

  • Consider including 0.3% hydrogen peroxide to block endogenous peroxidases

• Antibody concentration titration:

  • Test serial dilutions of primary antibody (1:100, 1:200, 1:500, 1:1000)

  • Optimize incubation time and temperature (overnight at 4°C often yields best results)

  • Consider signal amplification systems for low-abundance detection

• Counterstaining and controls:

  • Use mitochondrial markers (e.g., TOMM20) for co-localization confirmation

  • Include DAPI nuclear counterstain for cellular context

  • Run parallel negative controls (no primary antibody, isotype control)

This methodical approach helps achieve specific MRPS2 staining while minimizing background and false-positive signals.

How can I employ MRPS2 antibodies to study mitochondrial translation defects?

Investigating mitochondrial translation defects using MRPS2 antibodies requires sophisticated experimental approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use MRPS2 antibodies to pull down associated mitoribosomal components

  • Analyze protein complexes by mass spectrometry to identify abnormal interactions

  • Compare complex formation between healthy and disease models

  • Recommended buffer: 150mM NaCl, 50mM Tris-HCl pH 7.4, 1% NP-40, with protease inhibitors

• Sucrose gradient analysis:

  • Prepare mitochondrial lysates from cells/tissues

  • Separate on 10-30% sucrose gradients (centrifugation at 100,000g for 4 hours)

  • Collect fractions and analyze by Western blot with MRPS2 antibody

  • Assess ribosomal subunit assembly by comparing MRPS2 distribution across fractions

• Proximity labeling approaches:

  • Generate MRPS2-BioID or MRPS2-APEX2 fusion proteins

  • Identify proximal proteins in healthy vs. defective mitochondria

  • Validate interactions with MRPS2 antibody-based techniques

• Translation activity correlation:

  • Measure mitochondrial translation using 35S-methionine pulse labeling

  • Correlate translation activity with MRPS2 levels/localization

  • Perform rescue experiments with wild-type MRPS2 in defective models

• Super-resolution microscopy:

  • Utilize MRPS2 antibodies in STORM or STED microscopy

  • Examine spatial organization of mitochondrial translation machinery

  • Quantify changes in nanoscale distribution in disease states

These advanced techniques provide mechanistic insights into how MRPS2 dysfunction contributes to mitochondrial translation defects.

What approaches can resolve contradictory MRPS2 antibody results between different techniques?

When faced with contradictory results using MRPS2 antibodies across different techniques, implement the following systematic troubleshooting approach:

• Epitope accessibility assessment:

  • Different techniques expose different protein conformations

  • Western blotting: Denatured epitopes accessible

  • Immunohistochemistry/Immunofluorescence: Native/fixed epitopes accessible

  • Solution: Test antibodies targeting different regions of MRPS2 (N-terminal, C-terminal)

  • Consider epitope mapping to identify exactly which amino acids are recognized

• Cross-reactivity investigation:

  • Perform peptide competition assays with purified MRPS2 protein

  • Test antibody in MRPS2 knockout/knockdown models

  • Conduct immunoprecipitation followed by mass spectrometry to identify all bound proteins

  • Create a matrix correlating antibody performance across techniques:

• Fixation and preparation variables:

  • Systematically test multiple fixation protocols

  • Compare fresh vs. frozen vs. FFPE samples

  • Evaluate different antigen retrieval methods

  • Document all experimental conditions to identify critical variables

• Orthogonal validation approaches:

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Use alternative detection methods (e.g., proximity ligation assay)

  • Generate epitope-tagged MRPS2 constructs for antibody-independent detection

  • Consider absolute quantification using recombinant protein standards

This comprehensive approach helps resolve technical contradictions and identifies the most reliable conditions for each experimental context.

How do post-translational modifications affect MRPS2 antibody recognition?

Post-translational modifications (PTMs) can significantly impact MRPS2 antibody recognition, creating important experimental considerations:

• Known PTMs affecting MRPS2:

  • Phosphorylation: May occur at serine/threonine residues

  • Acetylation: Potential modification at lysine residues

  • Ubiquitination: Can signal for protein degradation

  • These modifications can mask or alter epitopes recognized by antibodies

• Modification-sensitive detection strategies:

  • Use phosphatase treatment prior to Western blotting to remove phosphorylation

  • Compare reducing vs. non-reducing conditions to assess disulfide involvement

  • Test sample preparation with/without deubiquitinating enzymes

  • Consider generating modification-specific antibodies for specialized studies

• Experimental approach to assess PTM interference:

  • Compare antibody reactivity in samples treated with:

    • Phosphatase inhibitors (maintains phosphorylation)

    • HDAC inhibitors (maintains acetylation)

    • Proteasome inhibitors (accumulates ubiquitinated forms)

  • Examine mobility shifts in Western blots that may indicate modified forms

  • Use 2D gel electrophoresis to separate MRPS2 isoforms by charge and size

• Technical recommendations:

  • For phosphorylation studies: Include calyculin A or okadaic acid in lysis buffers

  • For acetylation detection: Add trichostatin A during sample preparation

  • For detecting all forms: Use antibodies targeting regions less prone to modification

  • When possible, validate findings with mass spectrometry to identify specific modifications

Understanding the impact of PTMs on antibody recognition is crucial for accurate interpretation of experimental data, particularly when studying regulatory mechanisms affecting MRPS2 function.

How can I address weak or inconsistent MRPS2 signal in Western blots?

Weak or inconsistent MRPS2 signals in Western blots can be systematically improved through the following optimization strategies:

• Sample preparation optimization:

  • Ensure complete protein extraction with appropriate lysis buffers

  • Add protease inhibitor cocktail to prevent degradation

  • Consider mitochondrial enrichment to concentrate MRPS2 protein

  • Avoid repeated freeze-thaw cycles of protein samples

  • Verify protein concentration using reliable methods (BCA assay)

• Technical parameter adjustment:

  • Increase protein loading (up to 50-75μg per lane)

  • Optimize primary antibody concentration (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation time (up to 48 hours at 4°C)

  • Switch membrane type (PVDF may give better results than nitrocellulose)

  • Reduce washing stringency (lower salt concentration in wash buffer)

• Signal enhancement strategies:

  • Use high-sensitivity ECL substrates

  • Consider signal amplification systems (biotin-streptavidin)

  • Try HRP-conjugated MRPS2 antibody for direct detection

  • Optimize exposure time during imaging

  • Use cooled CCD camera systems for digital capture of weak signals

• Antibody selection considerations:

  • Test alternative MRPS2 antibodies targeting different epitopes

  • Compare polyclonal vs. monoclonal antibodies (polyclonals often provide stronger signals)

  • Verify antibody quality through recombinant protein detection

  • Consider lot-to-lot variation in antibody performance

• Positive control implementation:

  • Include samples known to express high levels of MRPS2

  • Use recombinant MRPS2 protein as a reference standard

  • Run housekeeping protein controls to verify equal loading

  • Document experimental conditions that produce consistent results

This comprehensive troubleshooting approach addresses the most common causes of weak or inconsistent MRPS2 signals in Western blotting.

What quality control measures should be implemented when using MRPS2 antibodies in publication-grade research?

For publication-grade research using MRPS2 antibodies, implement these rigorous quality control measures:

• Pre-experimental validation:

  • Verify antibody specificity through knockout/knockdown controls

  • Test antibody recognition of recombinant MRPS2 protein

  • Document antibody lot number, catalog number, and vendor

  • Perform titration experiments to determine optimal concentration

  • Conduct cross-reactivity testing against related mitochondrial ribosomal proteins

• Experimental controls:

  • Include positive controls (tissues/cells with confirmed MRPS2 expression)

  • Run negative controls (tissues/cells lacking MRPS2 expression)

  • Use technical replicates (minimum 3) to assess reproducibility

  • Include loading controls appropriate for the subcellular fraction

  • For mitochondrial proteins, consider VDAC or COX IV as loading controls

• Quantification and statistical analysis:

  • Normalize MRPS2 signals to appropriate loading controls

  • Present data from multiple independent experiments (n≥3)

  • Apply appropriate statistical tests based on data distribution

  • Report effect sizes alongside p-values

  • Include representative images alongside quantitative data

• Documentation and reporting:

  • Create detailed tables of antibody characteristics:

• Report detailed methodologies including blocking conditions, incubation times/temperatures

• Disclose any image processing or quantification methods

• Include supplementary validation data if space-limited in main manuscript

• Antibody validation reporting:

  • Follow antibody validation guidelines from journals and societies

  • Document multiple antibody validation approaches used

  • Share original, unmodified blot/image data in supplementary materials

  • Consider publicly sharing validation data through repositories

These quality control measures ensure experimental rigor and reproducibility when using MRPS2 antibodies in scientific publications.

How can MRPS2 antibodies be utilized in studying mitochondrial disease mechanisms?

MRPS2 antibodies offer valuable tools for investigating mitochondrial disease mechanisms through these advanced approaches:

• Diagnostic biomarker development:

  • Quantify MRPS2 levels in patient tissues/cells vs. healthy controls

  • Correlate MRPS2 abundance with mitochondrial translation efficiency

  • Assess MRPS2 incorporation into mitoribosomal complexes

  • Develop immunoassays for detecting abnormal MRPS2 expression/localization

• Pathophysiological mechanism elucidation:

  • Use MRPS2 antibodies to assess mitoribosome integrity in disease models

  • Perform proximity labeling to identify altered MRPS2 protein interactions

  • Correlate mitochondrial translation defects with MRPS2 dynamics

  • Implement time-course studies to track disease progression at molecular level

• Therapeutic response monitoring:

  • Evaluate MRPS2 expression/localization changes during treatment

  • Assess normalization of mitoribosome assembly following intervention

  • Develop companion diagnostic applications using MRPS2 antibodies

  • Create quantitative assays correlating MRPS2 status with clinical outcomes

• Genetic variant characterization:

  • Analyze impact of MRPS2 mutations on protein expression/stability

  • Compare mutant vs. wild-type MRPS2 localization using immunofluorescence

  • Assess functional consequences using antibody-based functional assays

  • Validate pathogenicity of variants of uncertain significance

• Mitochondrial quality control pathway investigation:

  • Track MRPS2 turnover under normal vs. stressed conditions

  • Study MRPS2 involvement in mitochondrial unfolded protein response

  • Examine MRPS2 fate during mitophagy using co-localization studies

  • Assess MRPS2 as a marker for mitochondrial homeostasis

These applications demonstrate how MRPS2 antibodies can advance understanding of fundamental disease mechanisms and potentially inform therapeutic approaches for mitochondrial disorders.

What techniques combine MRPS2 antibodies with advanced imaging methods for mitochondrial research?

Integration of MRPS2 antibodies with cutting-edge imaging technologies enables powerful new approaches to mitochondrial research:

• Super-resolution microscopy applications:

  • STED microscopy: Achieves ~30-50nm resolution to visualize individual mitoribosomes

  • STORM/PALM: Single-molecule localization microscopy reveals MRPS2 nanoscale organization

  • SIM: Structured illumination provides ~100nm resolution for mitoribosome distribution studies

  • Implementation protocol: Use highly specific primary MRPS2 antibodies with minimal cross-reactivity, followed by fluorophore-conjugated secondary antibodies optimized for super-resolution techniques

• Live-cell imaging strategies:

  • Combine mini-antibody fragments (Fab, nanobodies) against MRPS2 with cell-penetrating peptides

  • Implement SNAP/CLIP-tag labeling of MRPS2 for pulse-chase dynamics

  • Use split-GFP complementation to visualize MRPS2 interactions in living cells

  • Track mitoribosome assembly/disassembly in real-time during cellular stress

• Correlative light and electron microscopy (CLEM):

  • Localize MRPS2 via immunofluorescence, then examine ultrastructure of same region

  • Use immunogold labeling with MRPS2 antibodies for precise electron microscopy localization

  • Implement cryo-CLEM to preserve native structures during MRPS2 visualization

  • Quantify spatial relationships between mitoribosomes and other mitochondrial structures

• Expansion microscopy protocols:

  • Physically expand samples 4-10x using polyelectrolyte hydrogels

  • Use standard confocal microscopy to achieve effective super-resolution imaging of MRPS2

  • Perform multiplexed immunolabeling to examine MRPS2 in context of other mitochondrial components

  • Recommended protocol: Post-fixation immunolabeling using MRPS2 antibodies at 1:100-1:200 dilution

• Volumetric imaging approaches:

  • Implement tissue clearing techniques (CLARITY, iDISCO) compatible with MRPS2 immunolabeling

  • Perform light-sheet microscopy to visualize MRPS2 distribution throughout intact tissues

  • Create 3D reconstructions of mitochondrial networks with MRPS2 localization

  • Quantify spatial heterogeneity of MRPS2 expression across different cellular regions