MRPS12 Antibody

What is MRPS12 and why is it significant in mitochondrial research?

MRPS12 (Mitochondrial Ribosomal Protein S12) is an integral component of the small 28S subunit of mitochondrial ribosomes, essential for effective mitochondrial protein synthesis . The protein belongs to the ribosomal protein S12P family and serves as a key component of the ribosomal small subunit . Its significance stems from its crucial role in controlling decoding fidelity during mitochondrial translation and regulating susceptibility to aminoglycoside antibiotics . MRPS12 is encoded by nuclear genes rather than mitochondrial DNA, and the resulting protein is transported to mitochondria to participate in the assembly of mitoribosomes . Recent research has demonstrated that mutations in MRPS12 can significantly affect mitochondrial translation accuracy, with downstream effects on respiratory complex assembly and cellular metabolism .

What criteria should guide selection of an MRPS12 antibody for specific experimental applications?

When selecting an MRPS12 antibody, consider these research-specific factors:

For experimental rigor, select antibodies with documented validation in your specific application and cell/tissue type . Consider the immunogen used for antibody production—some MRPS12 antibodies are raised against full-length recombinant protein , while others target specific peptide sequences . This affects epitope accessibility in different applications and under various sample preparation conditions.

How do different host species and clonality options affect MRPS12 antibody performance?

The choice between rabbit, mouse, or other host species antibodies impacts experimental design in several ways:

Mouse polyclonal antibodies (e.g., ab167590) are suitable for Western blot applications with human samples, typically used at 1:500 dilution . These demonstrate good reactivity with MRPS12-transfected cell lysates.

Rabbit polyclonal antibodies (e.g., 15225-1-AP) offer broader application potential, including both Western blot (1:500-1:2000) and immunohistochemistry (1:50-1:500) . These antibodies have been validated in multiple human cell lines and tissue samples.

The clonality choice affects:

• Epitope recognition: Polyclonal antibodies recognize multiple epitopes on MRPS12, potentially increasing detection sensitivity but with higher risk of cross-reactivity .

• Batch consistency: While monoclonal antibodies offer better lot-to-lot consistency, the polyclonal antibodies currently available for MRPS12 have demonstrated reliable performance across multiple studies .

For co-staining experiments, consider host species compatibility with other primary antibodies to avoid cross-reactivity during secondary antibody detection.

What are the optimal protocols for Western blot detection of MRPS12?

For successful Western blot detection of MRPS12, follow this optimized protocol based on validated research approaches:

Sample Preparation:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Quantify protein concentration (BCA or Bradford assay)

• Load 15-20 μg of total protein per lane for most cell types

Gel Electrophoresis and Transfer:

• Use 12-15% SDS-PAGE gels to properly resolve the 13-15 kDa MRPS12 protein

• Transfer to PVDF membrane (preferred over nitrocellulose for small proteins)

• For HEK-293, HeLa, HepG2, or Jurkat cells, validated protocols consistently detect the expected band

Antibody Incubation:

• Block membrane with 5% non-fat milk in TBST for 1 hour

• Incubate with primary MRPS12 antibody diluted 1:500-1:2000 in blocking buffer overnight at 4°C

• Wash 3× with TBST

• Incubate with HRP-conjugated secondary antibody (1:2500-1:5000)

• Develop using enhanced chemiluminescence

Expected Results:
MRPS12 typically appears as a distinct band between 13-15 kDa . Transfected cells expressing MRPS12 show stronger signal intensity compared to non-transfected controls . Testing has confirmed successful detection in human samples with antibodies from multiple vendors .

How should I design immunohistochemistry experiments for MRPS12 detection in tissue samples?

For optimal immunohistochemical detection of MRPS12 in tissue samples:

Tissue Preparation:

• Fix tissue sections in 10% neutral buffered formalin and embed in paraffin

• Cut 4-6 μm sections and mount on positively charged slides

• Deparaffinize and rehydrate sections using standard protocols

Antigen Retrieval (Critical Step):

• Use TE buffer pH 9.0 for optimal epitope exposure

• Alternative: citrate buffer pH 6.0 may be used, though potentially with lower signal intensity

• Heat-induced epitope retrieval (pressure cooker or microwave) is recommended

Staining Protocol:

• Block endogenous peroxidase with 3% H₂O₂

• Apply protein block (serum-free) for 10 minutes

• Incubate with primary MRPS12 antibody at 1:50-1:500 dilution

• Apply HRP-polymer detection system

• Develop with DAB and counterstain with hematoxylin

Validated Tissues:
Human liver tissue has been extensively validated for MRPS12 detection . When examining other tissues, appropriate positive controls should be included alongside experimental samples.

Controls:
Include both positive control (human liver tissue) and negative control (primary antibody omitted) sections in each experimental run to validate staining specificity.

What controls and validation steps are essential when working with MRPS12 antibodies?

Implementing rigorous controls is critical for generating reliable data with MRPS12 antibodies:

Positive Controls:

• Cell lines: HEK-293, HeLa, HepG2, and Jurkat cells have validated MRPS12 expression

• Tissue samples: Human liver tissue shows consistent MRPS12 expression

• Overexpression systems: MRPS12-transfected 293T cells provide strong positive signal

Negative Controls:

• Primary antibody omission control to assess secondary antibody specificity

• Non-transfected cell lines as comparison for overexpression studies

• Competing peptide blocking to confirm epitope specificity

Validation Approaches:

• Molecular weight verification: Confirm detection at the expected 13-15 kDa band size

• siRNA knockdown: Demonstrate signal reduction following MRPS12 silencing

• Multiple antibody validation: Compare results using antibodies targeting different MRPS12 epitopes

• Cross-species reactivity assessment: Test antibody performance across human and mouse samples when conducting comparative studies

Documentation Requirements:
For publication-quality data, document complete antibody information including catalog number, dilution, incubation conditions, and lot number to ensure experimental reproducibility.

How can MRPS12 antibodies be used to investigate mitochondrial translation fidelity?

MRPS12 antibodies serve as powerful tools for investigating mitochondrial translation fidelity through several advanced approaches:

Pulse-Chase Analysis:

• Use MRPS12 antibodies to immunoprecipitate mitochondrial ribosomes following pulse-chase labeling with [³⁵S]-methionine

• Compare translation rates and newly synthesized protein stability between wild-type and MRPS12 mutant samples

• Research has shown that error-prone (ep) mutations in MRPS12 increase translation rates but reduce protein stability, while hyper-accurate (ha) mutations show different effects

Aminoglycoside Sensitivity Studies:
MRPS12 antibodies can help monitor how aminoglycosides like gentamicin affect mitochondrial translation in different genetic backgrounds. Studies have demonstrated that gentamicin treatment exacerbates protein instability in error-prone MRPS12 mutants, confirming increased amino acid misincorporation .

Respiratory Complex Assembly Analysis:

• Use MRPS12 antibodies alongside antibodies against mitochondria-encoded proteins to examine respiratory complex assembly

• Research reveals decreased assembly rates in Mrps12ep/ep mice despite maintained steady-state levels, indicating compensatory mechanisms

• BN-PAGE combined with pulse labeling can detect changes in assembly kinetics that steady-state measurements might miss

These approaches have revealed that MRPS12 mutations affecting translation fidelity result in tissue-specific phenotypes, with differential effects observed in heart versus liver tissue .

What role does MRPS12 play in metabolic stress responses and how can this be studied?

MRPS12's involvement in metabolic stress responses can be investigated using antibody-based approaches combined with metabolic manipulations:

High-Fat Diet Studies:
Research shows that metabolic stress modulates the effects of MRPS12 mutations. Mrps12ep/ep mice demonstrated different responses to normal chow diet versus high-fat feeding, suggesting MRPS12's role in adapting mitochondrial function to nutrient availability .

Tissue-Specific Effects Analysis:

• Use MRPS12 antibodies for comparative studies across tissues (particularly heart vs. liver)

• Research indicates that Mrps12ep/ep mice were protected against heart defects despite showing mitochondrial translation abnormalities

• This differential response suggests tissue-specific mechanisms that can be further investigated using MRPS12 antibodies in combination with markers of metabolic stress

Signaling Pathway Investigation:

• Combine MRPS12 antibodies with antibodies against stress pathway components (AKT, mTOR, S6)

• Studies found that phosphorylated S6 was decreased in both heart and liver of Mrps12ha/ha mice

• The mTOR target S6 and its phosphorylated form showed alterations in Mrps12 mutants, suggesting involvement of this pathway in responding to translation fidelity changes

Mitochondrial Stress Response Profiling:
MRPS12 antibodies can be used alongside markers of mitochondrial stress (CHOP, ATF4, TFAM) to determine how translation fidelity affects mitochondrial homeostasis. Research shows differential expression of these markers between error-prone and hyper-accurate MRPS12 mutants .

How can proteomic approaches be integrated with MRPS12 antibody applications?

Integrating proteomic approaches with MRPS12 antibody applications provides deeper insights into mitochondrial biology:

Co-Immunoprecipitation (Co-IP) Studies:

• Use MRPS12 antibodies to pull down intact mitochondrial ribosome complexes

• Mass spectrometry analysis of co-precipitated proteins reveals interaction partners

• This approach can identify differences in ribosome composition between wild-type and mutant MRPS12 conditions

Proximity Labeling Techniques:

• Create MRPS12-BioID or APEX2 fusion constructs

• Use MRPS12 antibodies to verify expression and localization of the fusion protein

• Identify proteins in proximity to MRPS12 under different metabolic conditions to map dynamic interaction networks

Quantitative Proteomics:
Proteomic analyses of heart mitochondria from Mrps12ha/ha mice revealed decreased levels of respiratory chain complex subunits . Similar approaches can be applied to other experimental models by using:

• SILAC labeling to quantify protein abundance changes

• MRPS12 antibodies for Western blot validation of key findings

• Pathway analysis to identify most affected biological processes

Post-Translational Modification Analysis:

• Immunoprecipitate MRPS12 under various stress conditions

• Use mass spectrometry to identify condition-specific post-translational modifications

• Generate modification-specific antibodies for further functional studies

This integrated approach has revealed that proteins involved in cardiac and mitochondrial metabolic processes were most affected by MRPS12 mutations .

What are common challenges in MRPS12 antibody applications and how can they be addressed?

When working with MRPS12 antibodies, researchers may encounter several technical challenges:

Multiple Bands in Western Blot:

• Cause: MRPS12 has multiple splice variants, though all encode the same protein . Additional bands may represent post-translational modifications or degradation products.

• Solution: Include positive control lysates (e.g., MRPS12-transfected 293T cells) to identify the correct band. Optimize sample preparation to minimize protein degradation by using fresh protease inhibitors and maintaining samples at 4°C.

Weak or No Signal in Immunohistochemistry:

• Cause: Inadequate antigen retrieval, particularly important for mitochondrial proteins.

• Solution: Test both recommended retrieval methods: TE buffer pH 9.0 (preferred) and citrate buffer pH 6.0 . Extend antigen retrieval time and optimize antibody concentration (try 1:50 dilution initially).

Cross-Reactivity with Non-Target Proteins:

• Cause: Some polyclonal antibodies may recognize epitopes shared with other proteins.

• Solution: Validate specificity using multiple antibodies targeting different MRPS12 epitopes. Consider using knockout or knockdown controls if available.

Variability Between Experiments:

• Cause: Antibody lot variation, storage degradation, or fluctuating experimental conditions.

• Solution: Note lot numbers, aliquot antibodies to avoid freeze-thaw cycles, and standardize protocols. Store according to manufacturer recommendations (-20°C, avoid repeated freeze/thaw cycles) .

How should researchers interpret MRPS12 expression patterns across different cell types and tissues?

Proper interpretation of MRPS12 expression patterns requires understanding its biological context:

Tissue-Specific Expression Patterns:

• High expression: MRPS12 is highly expressed in metabolically active tissues like heart and liver .

• Expression variability: Normal variation across tissues reflects different mitochondrial content and translational requirements.

• Interpretation approach: Always compare to appropriate tissue-matched controls rather than absolute expression levels.

Subcellular Localization:

• Expected localization: Primarily mitochondrial , appearing as punctate staining in immunofluorescence.

• Unexpected cytoplasmic staining: May indicate issues with mitochondrial import machinery or antibody specificity.

• Verification method: Co-stain with established mitochondrial markers (TOM20, MitoTracker) to confirm proper localization.

Disease State Considerations:

• Research has shown that MRPS12 function can be affected by metabolic disease states .

• In model systems, MRPS12 mutations show tissue-specific phenotypes, with differential effects in heart versus liver .

• When analyzing pathological samples, consider that altered MRPS12 expression or localization may reflect adaptive responses rather than causative changes.

Quantification Approaches:
When quantifying MRPS12 expression:

• Normalize to appropriate loading controls (β-actin for total protein; TOM20 for mitochondrial fraction)

• Consider normalizing to mitochondrial mass markers when comparing tissues with different mitochondrial content

• Use multiple technical and biological replicates to account for natural variation

How can researchers determine if experimental conditions are affecting MRPS12 antibody performance?

To systematically evaluate whether experimental conditions are affecting MRPS12 antibody performance:

Antibody Validation Controls:

• Include known positive control samples in each experiment (e.g., HEK-293, HeLa, HepG2, or Jurkat cells)

• Test multiple antibody dilutions (prepare a dilution series from 1:500 to 1:2000 for Western blot)

• Assess batch-to-batch consistency by maintaining reference samples

Sample Preparation Assessment:

• Compare fresh versus frozen samples to evaluate stability

• Test different lysis buffers (RIPA vs. NP-40 vs. Triton X-100)

• Evaluate the impact of different proteases inhibitor cocktails

Protocol Optimization Matrix:
Create a systematic testing grid:

Epitope Accessibility Considerations:
Mitochondrial proteins may require special treatment to expose epitopes:

• For fixed samples, test different fixatives (PFA vs. methanol)

• For tissue sections, compare heat-induced vs. enzymatic antigen retrieval

• Consider native vs. denaturing conditions for applications like immunoprecipitation

By systematically testing these variables, researchers can optimize conditions for their specific experimental system and ensure consistent MRPS12 antibody performance.

How might MRPS12 antibodies contribute to understanding metabolic disease mechanisms?

MRPS12 antibodies offer valuable tools for investigating metabolic disease mechanisms through several innovative approaches:

Tissue-Specific Mitochondrial Translation Analysis:
Studies have shown that Mrps12ep/ep mice were protected against heart defects but showed liver abnormalities under metabolic stress conditions . MRPS12 antibodies can help reveal:

• How mitochondrial translation fidelity differs between tissues in metabolic disease models

• Whether tissue-specific post-translational modifications of MRPS12 contribute to differential responses

• The relationship between translation fidelity and organ-specific disease manifestations

Metabolic Stress Adaptation Mechanisms:
MRPS12 antibodies can track changes in mitochondrial ribosome composition and function during metabolic adaptation:

• Compare diabetic versus healthy tissue samples for MRPS12 expression and modification patterns

• Investigate how dietary interventions affect MRPS12-dependent translation

• Examine whether exercise training modulates MRPS12 function in muscle tissue

Aminoglycoside Toxicity Research:
MRPS12 is implicated in sensitivity to aminoglycoside antibiotics . Antibodies can help determine:

• How genetic variants in MRPS12 might predict individual susceptibility to aminoglycoside-induced side effects

• Whether metabolic disease states alter aminoglycoside sensitivity profiles

• Potential protective interventions that might preserve mitochondrial translation fidelity during antibiotic treatment

What emerging techniques could enhance MRPS12 antibody applications in mitochondrial research?

Several cutting-edge techniques can significantly expand the utility of MRPS12 antibodies in mitochondrial research:

Super-Resolution Microscopy:

• Use MRPS12 antibodies with techniques like STORM or PALM to visualize mitochondrial ribosome distribution at nanoscale resolution

• Track spatial organization of translation machinery within mitochondria under different metabolic conditions

• Correlate MRPS12 positioning with nascent protein synthesis sites

Live-Cell Imaging Approaches:

• Develop cell-permeable MRPS12 antibody fragments for live-cell applications

• Create MRPS12-specific nanobodies for real-time monitoring of mitochondrial translation dynamics

• Combine with mitochondrial targeted reporters to correlate translation events with functional outcomes

Single-Cell Analysis:

• Apply MRPS12 antibodies in single-cell proteomics workflows to assess cell-to-cell variation in mitochondrial translation capacity

• Correlate with single-cell transcriptomics to understand nuclear-mitochondrial communication

• Identify rare cell populations with distinct mitochondrial translation profiles in heterogeneous tissues

CRISPR-Based Approaches:

• Generate MRPS12 knockin cell lines with epitope tags for enhanced antibody detection

• Create cellular models with MRPS12 mutations matching those found in patient populations

• Use MRPS12 antibodies to validate editing efficiency and study resulting phenotypes

By integrating these advanced techniques with established MRPS12 antibody applications, researchers can gain unprecedented insights into mitochondrial translation dynamics and their roles in health and disease.