MRPS31 Antibody

What are the typical specifications of commercially available MRPS31 antibodies?

Most commercially available MRPS31 antibodies are polyclonal antibodies produced in rabbits, with IgG isotype. Based on current market offerings, these antibodies typically demonstrate reactivity to human and rat MRPS31 proteins . They are generally available in unconjugated form for various applications:

What immunogens are typically used to produce MRPS31 antibodies?

Commercial MRPS31 antibodies are generally produced using one of two approaches:

• Recombinant fusion proteins: Most common is the recombinant fusion protein containing amino acids 66-395 of human MRPS31 (NP_005821.2) .

• Synthetic peptides: Some antibodies are raised against synthetic peptides corresponding to specific regions, such as amino acids 34-83 or 219-247 of human MRPS31.

The choice of immunogen affects antibody specificity and applications. For instance, antibodies targeting amino acids 66-395 typically demonstrate wider application versatility, while those targeting smaller epitopes (like aa 34-83) may have more specific binding characteristics but potentially limited applications .

Why does MRPS31 show discrepancy between calculated and observed molecular weight?

Researchers should be aware that the observed molecular weight of MRPS31 in Western blotting (approximately 37 kDa) differs significantly from its calculated molecular weight (45 kDa) . This discrepancy is not uncommon in protein research and can be attributed to several factors:

• Post-translational modifications affecting protein mobility in SDS-PAGE

• Protein processing events such as cleavage of signal peptides

• Anomalous migration due to protein structure or amino acid composition

• Presence of different modified forms of the protein simultaneously

As explained in technical literature: "Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

How should researchers optimize Western blot protocols when using MRPS31 antibodies?

When performing Western blot analysis with MRPS31 antibodies, researchers should implement the following optimization strategies:

• Sample preparation:

  • Use cell lines with known MRPS31 expression (Jurkat, HeLa, HepG2, MCF-7, A431)

  • Include mitochondrial enrichment steps for increased sensitivity

  • Use appropriate lysis buffers that effectively extract mitochondrial proteins

• Gel electrophoresis:

  • Use gradient gels (8-16%) to better resolve proteins around 37 kDa (the observed weight of MRPS31)

  • Load positive controls with known MRPS31 expression patterns

• Primary antibody incubation:

  • Start with manufacturer-recommended dilutions (1:500-1:2000)

  • Perform titration experiments to determine optimal concentration

  • Incubate at 4°C overnight for best results

• Detection considerations:

  • Look for the 37 kDa band rather than the calculated 45 kDa band

  • Include mitochondrial markers (e.g., VDAC, COX IV) as loading controls

  • Be aware that multiple bands may appear if different modified forms are present

Importantly, always verify specificity using appropriate controls and consider that observing multiple bands may not indicate non-specific binding but could represent different forms of the target protein.

What are the critical steps for successful immunofluorescence using MRPS31 antibodies?

For successful immunofluorescence (IF) detection of MRPS31, researchers should consider the following experimental steps:

• Cell preparation:

  • U2OS cells have been verified for IF applications with MRPS31 antibodies

  • Grow cells on glass coverslips to 60-80% confluence

  • Use freshly prepared cells for optimal results

• Fixation and permeabilization:

  • Test both paraformaldehyde (4%) and methanol fixation methods

  • For mitochondrial proteins, mild permeabilization with 0.1-0.2% Triton X-100 is typically sufficient

  • Overfixation may mask epitopes; optimize fixation time (typically 10-15 minutes)

• Antibody incubation:

  • Use dilutions in the recommended range (1:50-1:200 for IF)

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

  • Include control staining with mitochondrial markers (MitoTracker, TOM20) to confirm mitochondrial localization

• Visualization and analysis:

  • Expect a mitochondrial staining pattern (punctate or reticular cytoplasmic structures)

  • Use confocal microscopy for better resolution of mitochondrial structures

  • Consider using super-resolution techniques for detailed localization studies

Given that MRPS31 has cellular localization in the mitochondrion , colocalization with mitochondrial markers should be observed and can serve as an internal control for antibody specificity.

How does MRPS31 expression relate to hepatocellular carcinoma progression?

Research has revealed a significant relationship between MRPS31 loss and hepatocellular carcinoma (HCC) progression. Key findings include:

The research conclusively states that "MRPS31 loss is a key driver of mitochondrial deregulation and HCC aggressiveness," suggesting its potential as a novel biomarker for disease progression and patient stratification .

What molecular mechanisms link MRPS31 to cancer cell invasiveness?

MRPS31 influences cancer cell invasiveness through distinct molecular mechanisms depending on cancer type:

In hepatocellular carcinoma (HCC):

• MRPS31 deficiency disturbs mitoribosome assembly, leading to OXPHOS defects

• This deficiency enhances cell invasiveness through increased expression of MMP7 and COL1A1

• MMP7 contributes to extracellular matrix destruction

• COL1A1 modulates invasiveness via ZEB1-mediated epithelial-to-mesenchymal transition (EMT)

In breast cancer:

• MRPS31 is overexpressed in metastatic cell lines (MDA-MB231, MDA-MB468)

• It interacts with ACADSB, which is involved in fatty acid oxidation and has been associated with EMT

• In triple-negative breast cancer, MRPS31 also interacts with CES1 and NPAS2, both implicated in cancer progression

• MRPS31 has been associated with multiple cancer-related pathways including PIP3/AKT, hedgehog signaling, and Wnt signaling

These findings suggest that MRPS31 may play different—even opposing—roles depending on cancer type: loss promotes aggressiveness in HCC, while overexpression correlates with metastatic potential in breast cancer .

How does MRPS31 deficiency affect mitoribosome assembly and function?

MRPS31 deficiency has profound effects on mitoribosome assembly and function. Studies using hepatoma cell lines with SCNA-dependent MRPS31 expression (JHH5, HepG2, Hep3B, and SNU449) have demonstrated that:

• MRPS31 deficiency is a key mechanism disturbing the whole mitoribosome assembly .

• This disruption in mitoribosome assembly leads to:

  • Impaired mitochondrial protein synthesis

  • Defects in oxidative phosphorylation (OXPHOS)

  • Altered cellular metabolism

  • Potential metabolic reprogramming favoring cancer progression

• The impact of MRPS31 deficiency appears to be specific and not compensated by other mitoribosomal proteins, highlighting its essential role in mitoribosome structure and function .

These findings suggest that MRPS31 is not merely a structural component but plays a crucial functional role in mitoribosome integrity. The connection between mitoribosome dysfunction and cancer aggressiveness points to the importance of mitochondrial translation in maintaining cellular homeostasis and preventing disease progression.

What experimental approaches can be used to study MRPS31 copy number alterations?

Researchers investigating MRPS31 copy number alterations can employ several methodologies:

• Quantitative PCR (qPCR) for copy number analysis:

  • Use primers targeting MRPS31 genomic regions:

    • Region 1: 5′-GTGTTCTTGGTTCATTTCGTG and 5′-GACTATCAAATAAATCTTGGGAG

    • Region 2: 5′-ATGCCTACAATCCTAGCTGC and 5′-CTCAGTTCGAACAGACTGC

  • Include reference genes such as RB1 for normalization

• Fluorescence in situ hybridization (FISH):

  • Use probes targeting the MRPS31 locus on chromosome 13

  • Dual-color FISH with chromosome 13 centromere probe for aneusomy detection

• Array-comparative genomic hybridization (aCGH):

  • Genome-wide analysis of copy number alterations

  • Can identify co-occurring alterations on chromosome 13q along with MRPS31

• Next-generation sequencing approaches:

  • Whole genome sequencing for comprehensive copy number profiling

  • Targeted sequencing panels focusing on mitochondrial ribosomal proteins

• Digital droplet PCR (ddPCR):

  • Provides absolute quantification of copy number

  • Higher precision for detecting subtle copy number changes

For clinical correlations, researchers can stratify patient samples into MRPS31_high or MRPS31_low groups based on DNA copy number values (using thresholds of >0.2 or < −0.2 for the log2 transformed DCN value) . This stratification enables analysis of differential SCNA frequency patterns between groups and correlation with clinical outcomes.

How can researchers investigate the functional consequences of MRPS31 alterations in mitochondrial biology?

To investigate the functional impact of MRPS31 alterations on mitochondrial biology, researchers should consider these methodological approaches:

• Mitoribosome assembly analysis:

  • Sucrose gradient sedimentation to separate ribosomal subunits

  • Western blotting of fractions for mitoribosomal proteins

  • Analysis of 28S and 39S subunit formation

• Mitochondrial protein synthesis assays:

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

  • Analysis of newly synthesized mitochondrially-encoded proteins

• Oxidative phosphorylation assessment:

  • Oxygen consumption rate measurements (Seahorse XF Analyzer)

  • Analysis of individual respiratory complex activities

  • ATP production assays and membrane potential measurements

• Genetic manipulation of MRPS31:

  • CRISPR-Cas9 knockout or knockdown studies

  • Rescue experiments with wild-type MRPS31

  • Creation of cell lines modeling specific MRPS31 alterations found in cancer

• Proteomic analysis:

  • Mass spectrometry to identify altered mitochondrial proteome

  • Analysis of changes in mitoribosome composition

  • Investigation of protein-protein interactions involving MRPS31

• Metabolomic profiling:

  • Analysis of TCA cycle intermediates

  • Assessment of metabolic shifts (glycolysis vs. oxidative phosphorylation)

  • Evaluation of amino acid metabolism changes

These approaches can be particularly informative when applied to cell lines with documented SCNA-dependent MRPS31 expression (JHH5, HepG2, Hep3B, and SNU449) , providing relevant model systems for studying the consequences of MRPS31 alterations in cancer.

What strategies can be employed to investigate MRPS31 interactions with other proteins?

Understanding MRPS31's protein-protein interactions is crucial for elucidating its functions beyond mitoribosome structure. Researchers can implement these methodological approaches:

• Co-immunoprecipitation (Co-IP) with MRPS31 antibodies:

  • Utilize available MRPS31 antibodies for protein complex pulldown

  • Optimize buffer conditions to maintain physiologically relevant interactions

  • Identify interacting partners through mass spectrometry

• Proximity labeling approaches:

  • Create MRPS31 fusion constructs with BioID or APEX2

  • Express in relevant cell lines and activate the labeling enzyme

  • Identify labeled proximal proteins by mass spectrometry

• Cross-linking mass spectrometry:

  • Use reversible cross-linkers to stabilize transient interactions

  • Identify cross-linked peptides to map interaction interfaces

  • Particularly useful for studying components within ribosomal complexes

• Fluorescence resonance energy transfer (FRET):

  • Create fluorescent protein fusions with MRPS31 and candidate partners

  • Quantify energy transfer as an indicator of protein proximity

  • Live cell imaging to capture dynamic interactions

• Yeast two-hybrid screening:

  • Use MRPS31 as bait to identify novel interacting proteins

  • Validate identified interactions in mammalian systems

Based on existing research, priority should be given to investigating interactions with proteins involved in:

• Mitoribosome assembly and function

• Breast cancer metastasis pathways (ACADSB, CES1, NPAS2)

• Proteins involved in the epithelial-to-mesenchymal transition

• Components of the PIP3/AKT, hedgehog, and Wnt signaling pathways

How should researchers design experiments to evaluate MRPS31 as a potential biomarker in cancer?

To evaluate MRPS31's potential as a cancer biomarker, researchers should implement a comprehensive experimental design strategy:

Given MRPS31's differential roles in different cancer types (loss in HCC vs. overexpression in breast cancer metastasis) , cancer-specific validation is essential before clinical implementation.

What are the current limitations in MRPS31 research and future directions?

Current MRPS31 research faces several limitations that present opportunities for future investigations:

• Limited understanding of tissue-specific functions:

  • MRPS31 appears to have opposing roles in different cancers (loss in HCC vs. overexpression in breast cancer)

  • Future research should investigate tissue-specific functions and regulatory mechanisms

• Incomplete characterization of mitoribosome assembly:

  • While MRPS31 is known to be crucial for mitoribosome assembly , the precise molecular mechanisms remain unclear

  • Structural biology approaches could elucidate MRPS31's role in mitoribosome architecture

• Need for improved detection methods:

  • The discrepancy between calculated (45 kDa) and observed (37 kDa) molecular weight complicates analysis

  • Development of more specific antibodies and detection methods is needed

• Therapeutic targeting potential:

  • MRPS31's role in cancer progression suggests therapeutic potential

  • Future research should explore strategies to modulate MRPS31 function in disease contexts

• Expansion to other disease contexts:

  • MRPS31 has been associated with type 1 diabetes , but this relationship remains poorly understood

  • Investigation of MRPS31 in metabolic disorders and other diseases is warranted

• Regulatory mechanisms:

  • Understanding how MRPS31 expression and function are regulated under normal and pathological conditions

  • Exploring potential post-translational modifications affecting MRPS31 function