mrps-14 Antibody

What is MRPS14 and what cellular functions does it perform?

MRPS14 (mitochondrial ribosomal protein S14) is a 15 kDa protein component of the small mitochondrial ribosomal subunit. It plays a crucial role in mitochondrial protein synthesis by contributing to the assembly and stability of the mitochondrial ribosome. The protein is encoded by the nuclear genome but functions within mitochondria as part of the translation machinery that synthesizes proteins encoded by mitochondrial DNA . It should not be confused with MRP14 (also known as Calgranulin B or S100A9), which is an alarmin protein involved in inflammatory processes .

How is MRPS14 antibody different from MRP14/S100A9 antibody?

Despite their similar names, these antibodies target entirely different proteins with distinct functions:

MRPS14 antibody is primarily used to study mitochondrial ribosome structure and function, while MRP14 antibody is utilized for investigating inflammatory and immune responses .

What are the typical expression patterns of MRPS14 in different tissue types?

MRPS14 demonstrates relatively ubiquitous expression across multiple tissue types, reflecting the universal requirement for mitochondrial translation. Western blot analysis has confirmed MRPS14 expression in various human tissues including liver, stomach, and tonsil tissues. Additionally, it has been detected in several cell lines including RT4 and U251 MG cells . Unlike MRP14/S100A9, which is predominantly expressed in myeloid cells such as neutrophils and monocytes, MRPS14 expression correlates with mitochondrial content and activity across tissues .

What are the optimal sample preparation techniques for MRPS14 antibody applications?

For optimal results with MRPS14 antibody in various applications, the following preparation techniques are recommended:

Western Blot (WB):

• Use fresh tissue samples or cells lysed in a buffer containing protease inhibitors

• Recommended dilution: 1:500-1:1000 for MRPS14 antibody

• Sample denaturation at 95°C for 5 minutes in reducing buffer is essential for proper detection

• Include positive control samples such as mouse liver tissue

Immunohistochemistry (IHC):

• Formalin-fixed, paraffin-embedded (FFPE) tissues with appropriate antigen retrieval

• For optimal results, use TE buffer pH 9.0 for antigen retrieval (alternatively, citrate buffer pH 6.0)

• Recommended dilution: 1:50-1:500

• Blocking with 5% normal serum from the same species as the secondary antibody

Immunofluorescence (IF):

• Paraformaldehyde fixation (4%) followed by permeabilization with 0.1-0.5% Triton X-100

• Co-staining with mitochondrial markers can provide valuable context for localization studies

How can I validate MRPS14 antibody specificity for my experimental system?

Validating antibody specificity is critical for reliable research outcomes. For MRPS14 antibody, implement these validation strategies:

• Positive and negative controls:

  • Use known MRPS14-expressing tissues (mouse liver) as positive controls

  • Include samples with MRPS14 knockdown or knockout as negative controls

• Multiple detection methods:

  • Confirm findings using two different MRPS14 antibodies targeting different epitopes

  • Cross-validate with complementary techniques (e.g., mass spectrometry)

• Size verification:

  • Confirm the detected band matches the predicted molecular weight (15 kDa)

• siRNA knockdown:

  • Demonstrate reduced signal intensity following MRPS14-targeted siRNA treatment

  • Compare with non-targeting siRNA controls to confirm specificity

• Recombinant protein competition:

  • Pre-incubate antibody with purified MRPS14 protein before application

  • Signal reduction indicates specific binding to the target protein

What are the recommended dilutions and incubation conditions for different experimental applications?

The optimal working conditions for MRPS14 antibody applications are as follows:

It is strongly recommended to perform antibody titration for each experimental system to determine optimal conditions. Results can be sample-dependent, and optimization may be necessary for different tissue types or cell lines .

What are common causes of false-positive or false-negative results when using MRPS14 antibody?

False-positive results may occur due to:

• Cross-reactivity with similar proteins, particularly when using polyclonal antibodies

• Excessive antibody concentration causing non-specific binding

• Insufficient blocking leading to background signal

• Sample contamination with non-target proteins

• Inadvertent detection of MRP14/S100A9 instead of MRPS14 due to name confusion

False-negative results may be attributed to:

• Insufficient antigen retrieval for IHC applications

• Protein degradation during sample preparation

• Inefficient protein transfer in Western blot

• Epitope masking due to protein modifications or interactions

• Suboptimal storage conditions affecting antibody activity

• Inappropriate sample preparation methods for mitochondrial proteins

To mitigate these issues, always include appropriate controls, optimize protocols for mitochondrial protein detection, and verify results using complementary techniques.

How can I improve signal-to-noise ratio when using MRPS14 antibody for immunohistochemistry?

To enhance signal specificity while minimizing background in IHC applications:

• Optimize antigen retrieval:

  • Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine optimal conditions

  • Adjust retrieval time and temperature based on tissue type and fixation method

• Implement stringent blocking:

  • Use 5-10% normal serum from the secondary antibody species

  • Add 0.1-0.3% Triton X-100 to improve antibody penetration

  • Include BSA or non-fat dry milk to reduce non-specific binding

• Antibody optimization:

  • Titrate antibody concentration (starting with 1:50 dilution and testing up to 1:500)

  • Extend primary antibody incubation time (overnight at 4°C often yields better results)

  • Use diluents with protein carriers to maintain antibody stability

• Enhanced washing:

  • Increase washing duration and volume between antibody applications

  • Use gentle agitation during washing steps to remove unbound antibody

  • Add 0.05-0.1% Tween-20 to wash buffers to reduce background

• Signal amplification systems:

  • Consider using polymer-based detection systems for enhanced sensitivity with reduced background

  • Biotin-free detection systems eliminate endogenous biotin-related background

What considerations should be made when analyzing MRPS14 in tissues with varying mitochondrial content?

Mitochondrial content varies significantly across tissue types and physiological states, which impacts MRPS14 detection and interpretation:

• Normalization approaches:

  • Normalize MRPS14 levels to established mitochondrial markers (TOM20, VDAC, or Citrate Synthase)

  • Consider using mitochondrial DNA content as an additional normalization parameter

  • Compare ratios of MRPS14 to other mitochondrial ribosomal proteins rather than absolute values

• Tissue-specific considerations:

  • High mitochondrial content tissues (heart, liver, kidney) will naturally show stronger MRPS14 signals

  • Account for mitochondrial biogenesis fluctuations in response to stress or metabolic changes

  • Consider using mitochondrial isolation procedures for more accurate quantification

• Experimental controls:

  • Include tissues with known mitochondrial content differences as reference points

  • Utilize both mitochondrial and cytosolic housekeeping proteins as loading controls

  • Implement mitochondrial enrichment protocols for tissues with low mitochondrial content

How can MRPS14 antibody be used to investigate mitochondrial translation defects in disease models?

MRPS14 antibody serves as a valuable tool for exploring mitochondrial translation abnormalities in various pathological conditions:

• Ribosome assembly analysis:

  • Use sucrose gradient fractionation followed by Western blot with MRPS14 antibody to assess mitochondrial ribosome assembly

  • Compare small subunit incorporation patterns between healthy and disease models

  • Evaluate co-immunoprecipitation of MRPS14 with other ribosomal components

• Translational activity correlation:

  • Combine MRPS14 immunodetection with mitochondrial translation assays using radiolabeled amino acids

  • Assess whether MRPS14 levels correlate with translation efficiency in disease states

  • Investigate post-translational modifications of MRPS14 that might affect ribosome function

• Disease-specific applications:

  • In mitochondrial myopathies: Analyze MRPS14 localization and expression in muscle biopsies

  • In neurodegenerative disorders: Evaluate MRPS14 distribution in affected vs. unaffected brain regions

  • In cancer models: Assess MRPS14 alterations in relation to metabolic reprogramming

• Therapeutic response monitoring:

  • Use MRPS14 antibody to track mitochondrial recovery following treatment interventions

  • Monitor changes in mitochondrial ribosome composition during cellular stress responses

  • Evaluate drug effects on mitochondrial translation machinery

What techniques can be used to study MRPS14 interactions with other mitochondrial ribosomal proteins?

Investigating MRPS14's protein-protein interactions provides critical insights into mitochondrial ribosome assembly and function:

• Co-immunoprecipitation (Co-IP):

  • Use MRPS14 antibody for pull-down experiments followed by mass spectrometry

  • Perform reverse Co-IP with antibodies against known interacting partners

  • Implement crosslinking prior to Co-IP to capture transient interactions

• Proximity ligation assay (PLA):

  • Combine MRPS14 antibody with antibodies against potential interacting partners

  • Visualize and quantify specific interactions within subcellular compartments

  • Assess how pathological conditions affect interaction patterns

• FRET/FLIM analysis:

  • Utilize fluorescently-tagged antibodies to study protein proximity in fixed cells

  • Measure energy transfer between MRPS14 and putative interacting proteins

  • Detect conformational changes during ribosome assembly

• In silico structural analysis:

  • Correlate experimental findings with cryo-EM structures of mitochondrial ribosomes

  • Predict interaction interfaces based on evolutionary conservation patterns

  • Design targeted experiments to validate computational predictions

Published research has identified interactions between MRPS14 and other proteins, including OXA1L, which is involved in cotranslational quality control in mitochondria .

How can MRPS14 antibody be applied in studies investigating mitochondrial stress responses?

MRPS14 antibody applications in mitochondrial stress research include:

• Translational adaptation to stress:

  • Track MRPS14 localization and abundance changes during oxidative stress, hypoxia, or nutrient deprivation

  • Correlate alterations in MRPS14 with mitochondrial protein synthesis rates

  • Investigate post-translational modifications of MRPS14 under stress conditions

• Mitochondrial unfolded protein response (UPRmt):

  • Analyze MRPS14 redistribution during UPRmt activation

  • Assess relationships between MRPS14 levels and expression of UPRmt markers

  • Evaluate MRPS14 incorporation into ribosomes during recovery from proteotoxic stress

• Mitophagy monitoring:

  • Use MRPS14 as a marker for selective degradation of mitochondrial components

  • Compare clearance rates of MRPS14 versus outer membrane proteins during mitophagy

  • Investigate MRPS14 recycling mechanisms during mitochondrial turnover

• Multi-parametric analysis:

  • Combine MRPS14 immunodetection with membrane potential indicators

  • Correlate MRPS14 patterns with ROS production and ATP synthesis

  • Integrate MRPS14 data with transcriptomic and proteomic profiles of stress responses

How should researchers interpret changes in MRPS14 expression relative to other mitochondrial proteins?

Accurate interpretation of MRPS14 expression patterns requires nuanced contextual analysis:

• Coordinated vs. differential expression:

  • Determine whether MRPS14 changes parallel other mitochondrial ribosomal proteins

  • Assess whether MRPS14 alterations precede or follow changes in respiratory chain components

  • Evaluate stoichiometric relationships between small and large ribosomal subunit proteins

• Tissue-specific reference ranges:

  • Establish normal expression ranges for specific tissues or cell types

  • Consider developmental stage and metabolic state when interpreting expression data

  • Account for varying mitochondrial content across different tissue types

• Pathway integration:

  • Relate MRPS14 changes to mitochondrial biogenesis pathways (PGC1α, NRF1, TFAM)

  • Assess correlations with mitochondrial translation outputs

  • Evaluate relationships with cellular stress response mechanisms

• Functional correlation:

  • Connect MRPS14 expression patterns with functional readouts (respiration, ATP production)

  • Assess whether MRPS14 alterations cause or result from functional changes

  • Determine threshold levels required for normal mitochondrial translation

What are common methodological pitfalls when comparing MRPS14 antibody data from different experimental platforms?

Cross-platform data integration presents several challenges that researchers should address:

• Antibody variation:

  • Different antibodies may recognize distinct epitopes, affecting detection sensitivity

  • Polyclonal vs. monoclonal antibodies may yield different expression patterns

  • Batch-to-batch variation can impact quantitative comparisons

• Sample preparation differences:

  • Protein extraction methods significantly impact mitochondrial protein recovery

  • Fixation protocols for microscopy can alter epitope accessibility

  • Subcellular fractionation techniques vary in mitochondrial purity and yield

• Detection system variations:

  • Chemiluminescence vs. fluorescence-based Western blot detection affects dynamic range

  • Chromogenic vs. fluorescent IHC detection systems have different sensitivity thresholds

  • Digital vs. film-based image acquisition introduces systematic biases

• Quantification approaches:

  • Normalization strategies (total protein, housekeeping genes, mitochondrial markers) affect results

  • Image analysis algorithms may apply different background correction methods

  • Statistical treatments of semi-quantitative data require careful standardization

To mitigate these issues, implement standardized protocols, use the same antibody across experiments when possible, and include calibration standards for quantitative comparisons.

How can contradictory MRPS14 antibody results be reconciled in complex experimental systems?

When faced with contradictory results, employ these systematic reconciliation approaches:

• Technical validation:

  • Verify antibody specificity using knockout/knockdown controls

  • Test multiple antibodies targeting different epitopes

  • Confirm findings with orthogonal techniques (mRNA quantification, mass spectrometry)

• Biological complexity assessment:

  • Consider post-translational modifications that may affect epitope recognition

  • Evaluate protein complex formation that could mask antibody binding sites

  • Assess subcellular compartmentalization that might influence detection efficiency

• Experimental condition audit:

  • Catalog all experimental variables including sample preparation, buffer compositions, and incubation times

  • Standardize critical parameters across experimental platforms

  • Implement factorial design experiments to identify interacting variables

• Data integration framework:

  • Develop hierarchical models that incorporate reliability assessments for each data source

  • Apply Bayesian approaches to weight evidence based on methodological rigor

  • Use systems biology approaches to contextualize findings within broader networks

  • Conduct meta-analysis of similar experiments reported in the literature

Contradictory results often reveal important biological complexities rather than experimental failures, and their reconciliation can lead to novel insights into mitochondrial biology.

How might MRPS14 antibody applications evolve with advancements in single-cell and spatial proteomics?

Emerging technologies will transform MRPS14 research through:

• Single-cell resolution:

  • Application of MRPS14 antibodies in mass cytometry (CyTOF) for high-dimensional analysis

  • Adaptation for single-cell Western blot techniques to capture cell-to-cell variability

  • Integration with single-cell transcriptomics to correlate protein and mRNA levels

• Spatial proteomics advancements:

  • Implementation in multiplexed ion beam imaging (MIBI) to map MRPS14 distribution with nanometer precision

  • Application in Imaging Mass Cytometry to correlate MRPS14 with multiple other proteins simultaneously

  • Utilization in proximity extension assays for in situ detection of protein interactions

• Combined modality approaches:

  • Development of split-reporter systems based on MRPS14 antibody fragments

  • Creation of MRPS14-specific aptamer probes for live-cell applications

  • Integration with CRISPR-based tagging systems for endogenous protein tracking

These technological advances will enable unprecedented insights into mitochondrial heterogeneity and dynamic ribosome assembly processes at single-organelle resolution.

What role might MRPS14 antibodies play in understanding mitochondrial contributions to neurodegenerative diseases?

MRPS14 antibodies will be increasingly valuable for investigating mitochondrial dysfunction in neurodegeneration:

• Disease-specific applications:

  • Analysis of MRPS14 distribution in post-mortem brain tissues from patients with Alzheimer's, Parkinson's, and ALS

  • Evaluation of mitochondrial translation efficiency in patient-derived neurons and glial cells

  • Assessment of MRPS14 modifications (phosphorylation, acetylation) in disease progression

• Mechanistic investigations:

  • Tracking MRPS14-containing mitochondrial ribosomes during axonal transport

  • Evaluating regional variations in mitochondrial translation capacity across brain structures

  • Monitoring MRPS14 turnover in response to proteotoxic stress in neurons

• Therapeutic development:

  • Screening compounds that normalize MRPS14 incorporation into mitochondrial ribosomes

  • Monitoring mitochondrial translation recovery following experimental therapies

  • Developing MRPS14-based biomarkers for disease progression or treatment response

The relationship between mitochondrial translation defects and neurodegenerative pathologies represents a promising frontier for MRPS14 antibody applications.