MRPS5 Antibody

What is MRPS5 and what cellular functions does it perform?

MRPS5 (Mitochondrial Ribosomal Protein S5) is a structural protein that forms part of the 28S small subunit of the mitochondrial ribosome. It plays a crucial role in mitochondrial protein synthesis, specifically in the assembly of the mitochondrial ribosome, which is essential for the translation of proteins encoded by mitochondrial DNA. These proteins are primarily components of the electron transport chain involved in oxidative phosphorylation (OXPHOS) and ATP generation .

The mitochondrial ribosome, composed of a 28S small subunit and a 39S large subunit, is responsible for translating 13 specific proteins encoded by mitochondrial DNA. These proteins are integral to cellular energy metabolism as they form essential components of the mitochondrial electron transport chain . MRPS5 has also been identified as a longevity-related protein that can regulate lifespan through mitochondrial energy metabolism disorders .

For optimal detection of MRPS5 via Western blot, several critical steps should be followed:

• Lysate preparation: Total cell lysates from cell lines such as HeLa, Jurkat, HEK-293, or HepG2 have shown good results for MRPS5 detection. Standard lysis buffers containing protease inhibitors are recommended .

• Protein loading: Approximately 30 μg of protein per lane has been shown to be sufficient for detection .

• Gel selection: 10% SDS-PAGE gels are commonly used for separation of MRPS5, which has a predicted molecular weight of 48 kDa but is typically observed at 38-42 kDa .

• Transfer conditions: Standard semi-dry or wet transfer protocols are suitable.

• Blocking: 5% non-fat milk or BSA in TBST is typically used.

• Antibody incubation: Primary antibody dilutions of 1:500-1:3000 have been shown to be effective, with overnight incubation at 4°C yielding optimal results .

• Detection method: Both chemiluminescence and fluorescence-based detection systems are compatible.

It should be noted that the observed molecular weight (38-42 kDa) may differ from the calculated molecular weight (48 kDa) due to post-translational modifications or protein processing .

What controls should be used when working with MRPS5 antibodies?

Proper experimental controls are essential for validating MRPS5 antibody specificity and ensuring reliable results:

• Positive controls: Cell lines with confirmed MRPS5 expression such as HEK-293, HeLa, HepG2, and Jurkat cells have been validated as suitable positive controls .

• Negative controls:

  • Omission of primary antibody while maintaining all other steps

  • Use of isotype-matched control antibodies (rabbit IgG for polyclonal antibodies)

  • MRPS5 knockdown or knockout cells, if available

• Loading controls: Mitochondrial markers such as VDAC or COX IV can be used to normalize MRPS5 expression to mitochondrial content rather than total cellular protein.

• Subcellular fractionation control: When studying mitochondrial localization, proper fractionation should be validated using markers for mitochondria (VDAC), cytosol (GAPDH), and nuclear (Lamin B) compartments .

• Recombinant protein: Purified recombinant MRPS5 can serve as a definitive positive control for antibody validation .

How does MRPS5 expression correlate with disease prognosis in cancer studies?

Research has demonstrated that MRPS5 expression levels significantly correlate with prognosis in certain cancers, particularly clear cell renal cell carcinoma (ccRCC):

In ccRCC, MRPS5 expression was found to be downregulated in tumoral tissues compared to peritumoral tissues. This was confirmed by multiple detection methods including qRT-PCR, western blotting, and immunohistochemical staining .

A comprehensive study of 160 ccRCC patient samples revealed that:

• 61.9% (99 cases) showed high expression of MRPS5

• 38.1% (61 cases) showed low expression of MRPS5

The expression level of MRPS5 significantly correlated with several clinicopathological parameters:

• T classification (p=0.033)

• TNM stage (p=0.035)

• Fuhrman grade (p<0.001)

Patients with low MRPS5 expression demonstrated:

Multivariate analysis identified MRPS5 expression as an independent predictor of:

What mechanisms explain the role of MRPS5 in mitochondrial translation accuracy?

MRPS5 plays a critical role in maintaining the accuracy of mitochondrial translation, with mutations in this protein leading to mistranslation:

Experimental models using directed mutagenesis of MRPS5 have demonstrated that specific mutations can lead to mitoribosomal mistranslation. For example, the V336Y mutation in human MRPS5 corresponds to known ribosomal ambiguity mutations in homologous proteins and results in increased mistranslation .

The mechanism involves:

• Structural alterations: The V336Y mutation introduces a large aromatic tyrosine residue that causes steric hindrance within the C-terminal domain of MRPS5 .

• Measurable mistranslation effects:

  • Increased ratio of cysteine/methionine incorporation in mitochondrially-encoded proteins

  • Enhanced read-through of stop codons, resulting in extended proteins (particularly observed with MT-CO1 protein)

• Aminoglycoside sensitivity: Cells with MRPS5 mutations show heightened sensitivity to aminoglycoside-induced mistranslation, similar to cells carrying the pathogenic A1555G mutation in mitochondrial 12S rRNA .

• Reduced complex IV/complex II activity ratio in mitochondria

• Decreased ATP levels

• Increased production of mitochondrial reactive oxygen species (mtROS)

These findings establish MRPS5 as a critical determinant of mitochondrial translation accuracy and link mistranslation to mitochondrial dysfunction.

How can MRPS5 be manipulated to study cardiac regeneration mechanisms?

Recent research has revealed that MRPS5 manipulation represents a novel approach to study and potentially enhance cardiac regeneration:

A breakthrough study demonstrated that deletion of a single allele of MRPS5 in mice (Mrps5+/-) results in:

• Elevated cardiomyocyte proliferation

• Enhanced cardiac regeneration

• Improved cardiac function after myocardial infarction

The molecular mechanism involves:

• Mitochondrial stress response activation: Heterozygous loss of MRPS5 inhibits mitochondrial translation, creating mitonuclear protein imbalance .

• ATF4 upregulation: ATF4 (activating transcription factor 4) was identified as a key regulator of this mitochondrial stress response in cardiomyocytes from Mrps5+/- mice .

• Knl1 regulation: ATF4 regulates Knl1 (kinetochore scaffold 1), leading to an increase in cytokinesis during cardiomyocyte proliferation .

The importance of this pathway was confirmed through genetic studies:

• Deletion of one Atf4 allele in Mrps5+/- mice (creating Mrps5+/-/Atf4+/- double heterozygotes) attenuated cardiomyocyte proliferation

• This genetic modification also resulted in loss of cardiac regenerative capacity

This mechanism appears to be conserved across species, as MRPS5 inhibition or doxycycline treatment (which also inhibits mitochondrial translation) activated similar regulatory mechanisms in human induced pluripotent stem cell-derived cardiomyocytes, increasing their proliferation .

These findings have significant implications for understanding cardiac regeneration and developing potential therapeutic strategies for heart repair.

What role does MRPS5 play in mitonuclear communication during cardiac stress?

MRPS5 has emerged as a crucial regulator of mitonuclear communication—the informational flow from mitochondria to the nucleus—particularly during cardiac stress:

Comprehensive studies of MRPS5 function in cardiac development and disease have demonstrated that:

• Loss of Mrps5 in developing heart leads to cardiac defects and embryonic lethality

• Postnatal loss of Mrps5 induces cardiac hypertrophy and heart failure

The cellular consequences of MRPS5 deficiency in cardiomyocytes include:

• Disrupted mitochondrial structure and function

• Impaired mitochondrial protein translation

• Compromised oxidative phosphorylation (OXPHOS)

Mechanistic investigations revealed:

• Klf15 as a downstream target: Mrps5 regulates the expression of Krüppel-like factor 15 (Klf15)

• Rescue by Klf15: Exogenous Klf15 expression can rescue defects caused by Mrps5 deficiency and rebalance the cardiac metabolome

• Repression mechanism: Mrps5 represses Klf15 expression through c-myc, together with the metabolite L-phenylalanine

This intricate regulation of nuclear gene expression by a mitochondrial ribosomal protein illustrates how disruption of mitochondrial translation can trigger compensatory nuclear responses through metabolic signaling pathways.

The identification of this MRPS5-Klf15 axis in mitonuclear communication highlights potential therapeutic targets for heart failure and other conditions characterized by mitochondrial dysfunction .

How do genetic variants in MRPS5 contribute to disease susceptibility?

Genetic variations in MRPS5 have been associated with susceptibility to specific diseases, with detailed mechanisms beginning to emerge:

A rare missense variant in MRPS5, rs200730619 (c. 95108402T>C [p. Tyr137Cys]), was identified through whole exome sequencing as a contributing factor to leprosy risk:

• Validated in 369 cases and 270 controls of Chinese descent

• Showed significant association: P adjusted = 0.006, odds ratio (OR) = 2.74

Functional studies revealed that:

• mRNA levels of MRPS5 were downregulated in peripheral blood mononuclear cells stimulated with Mycobacterium leprae sonicate

• This suggests MRPS5 may be involved in the host immune response to the pathogen

The proposed mechanism involves:

• Defective MRPS5 potentially leading to impaired energy metabolism in host immune cells

• Resulting in defects in clearing M. leprae

• Ultimately increasing susceptibility to infection

Additionally, the pathogenic A1555G mutation in mitochondrial 12S rRNA, which causes maternally transmitted deafness, has mechanistic connections to MRPS5 function:

• Both A1555G mutation and MRPS5 mutations can cause mitoribosomal mistranslation

• They show similar sensitivity to aminoglycoside-induced translation errors

• This suggests common pathways through which different genetic variants can disrupt mitochondrial function

These findings illustrate how variations in mitochondrial ribosomal components can influence disease susceptibility through effects on protein translation, energy metabolism, and immune cell function.

What are the recommended storage and handling conditions for MRPS5 antibodies?

Proper storage and handling of MRPS5 antibodies are crucial for maintaining their performance and specificity over time:

Storage conditions:

• Most MRPS5 antibodies should be stored at -20°C

• They remain stable for one year after shipment when stored properly

• For antibodies supplied in small volumes (20 μl), aliquoting is generally unnecessary for -20°C storage

Storage buffer composition:

• Typically contains PBS with 0.02-0.03% sodium azide and 50% glycerol at pH 7.3-7.4

• Some formulations may contain 0.1% BSA as a stabilizer

Handling recommendations:

• Avoid repeated freeze-thaw cycles (more than 5)

• When removing from storage, thaw on ice or at 4°C

• Mix gently by inverting the tube several times rather than vortexing

• Briefly centrifuge before opening to collect solution at the bottom of the tube

• Keep on ice during experiments and return to -20°C promptly after use

• Wear gloves when handling to prevent contamination

Working solution preparation:

• Dilute only the amount needed for each experiment

• Use high-quality, freshly prepared buffers

• For Western blot applications, prepare dilutions in blocking buffer containing 5% non-fat milk or BSA in TBST

Following these recommendations will help ensure consistent antibody performance across experiments and maximize the useful life of the reagent.

How can researchers validate MRPS5 knockdown or knockout models?

Validating MRPS5 knockdown or knockout models requires a multi-faceted approach to confirm the success of genetic manipulation and assess functional consequences:

Molecular validation techniques:

• mRNA expression analysis:

  • RT-qPCR targeting MRPS5 transcript using validated primers

  • Normalized to stable reference genes (e.g., GAPDH, β-actin)

• Protein expression analysis:

  • Western blot using validated anti-MRPS5 antibodies (1:500-1:3000 dilution)

  • Immunofluorescence to assess mitochondrial localization and expression levels

• Genomic validation:

  • PCR of genomic DNA to confirm successful insertion of knockout cassette or CRISPR-induced mutations

  • Sequencing of targeted region to verify the precise genetic modification

Functional validation approaches:

• Mitochondrial translation assessment:

  • In organello translation assays using radiolabeled amino acids (35S-Met)

  • SDS-PAGE analysis of mitochondrially synthesized proteins

  • Measurement of translation accuracy via cysteine/methionine incorporation ratio

  • Assessment of read-through using extended protein products (e.g., MT-CO1)

• Mitochondrial function tests:

  • Oxygen consumption rate measurements

  • ATP level quantification

  • Mitochondrial reactive oxygen species (mtROS) detection

  • Complex activities (especially complex IV/complex II ratio)

• Cell proliferation and viability:

  • Growth curve analysis and generation time calculation

  • Viability assays (MTT, alamarBlue)

  • Cell cycle analysis

• Tissue-specific phenotypes:

  • For cardiac studies: echocardiography, histological assessment of heart structure

  • For other tissues: appropriate functional tests based on the tissue of interest

When establishing heterozygous models (e.g., MRPS5+/-), careful quantification of the degree of MRPS5 reduction is particularly important, as these models often show specific phenotypes distinct from complete knockout models .

How can mitochondrial translation be accurately measured in MRPS5 studies?

Accurate measurement of mitochondrial translation is essential when studying MRPS5 function or its manipulation. Several complementary approaches can be employed:

Radioactive labeling techniques:

• 35S-Methionine incorporation assay:

  • Requires inhibition of cytoplasmic translation (typically with cycloheximide or emetine)

  • Pulse-labeling with 35S-methionine for 30-60 minutes

  • Followed by SDS-PAGE separation and phosphorimaging or autoradiography

  • Allows quantification of total mitochondrial translation rate

  • Can be analyzed both quantitatively (total signal) and qualitatively (pattern of translated products)

• Pulse-chase analysis:

  • Extends the above method with a "chase" period using non-radioactive methionine

  • Enables assessment of protein stability and turnover rates

  • Critical for distinguishing translation defects from degradation effects

Non-radioactive methods:

• Click chemistry approaches:

  • Uses non-radioactive methionine analogs (e.g., L-azidohomoalanine, AHA)

  • Detection via copper-catalyzed azide-alkyne cycloaddition

  • Compatible with various detection systems including fluorescence and biotin-streptavidin

• Puromycin incorporation:

  • Nascent peptide chains incorporate puromycin at their C-termini

  • Detection via anti-puromycin antibodies

  • Useful for measuring active translation sites

Translation accuracy assessment:

• Amino acid misincorporation analysis:

  • Measuring cysteine/methionine incorporation ratio

  • Higher ratios indicate increased mistranslation

• Read-through assessment:

  • Detection of extended protein products (e.g., MT-CO1)

  • Increased with mistranslation

  • Can be enhanced by adding aminoglycosides like tobramycin

• Mass spectrometry:

  • Precise identification of mistranslated residues

  • Can provide comprehensive proteome-wide view of translation errors

Controls and considerations:

• Always include wild-type cells/tissues as positive controls

• For aminoglycoside studies, include dose-response curves

• Normalize to mitochondrial content using markers like VDAC

• Account for potential changes in mitochondrial mass when comparing samples

• Consider parallel assessment of cytoplasmic translation for comparison

These methods collectively provide a comprehensive assessment of both the quantity and quality of mitochondrial translation in the context of MRPS5 studies.

What are common issues with MRPS5 detection in Western blots and how can they be resolved?

Researchers may encounter several challenges when using MRPS5 antibodies for Western blot applications. Here are common issues and their solutions:

Problem: No or weak signal

Potential causes and solutions:

• Insufficient protein amount: Increase loading to 30-50 μg per lane

• Inefficient transfer: Optimize transfer conditions; consider extended transfer time for this 48 kDa protein

• Antibody concentration too low: Try higher primary antibody concentration (up to 1:500)

• Degraded antibody: Use fresh aliquot and verify storage conditions

• Low MRPS5 expression: Confirm expression in your sample type; use positive controls like HeLa or HEK-293 lysates

Problem: Multiple bands or unexpected molecular weight

Potential causes and solutions:

• Predicted vs. observed weight discrepancy: MRPS5 is calculated to be 48 kDa but often observed at 38-42 kDa; this is normal

• Non-specific binding: Increase blocking time/concentration; try different blocking reagents (milk vs. BSA)

• Cross-reactivity: Use higher antibody dilution (1:2000-1:3000); try different antibody clone

• Degradation products: Add fresh protease inhibitors; keep samples cold; reduce processing time

• Post-translational modifications: This could be biologically relevant; verify with additional antibodies

Problem: High background

Potential causes and solutions:

• Insufficient blocking: Increase blocking time to 1-2 hours; try 5% BSA instead of milk

• Antibody concentration too high: Dilute primary antibody further (1:2000-1:3000)

• Insufficient washing: Increase number and duration of wash steps

• Membrane issues: Use fresh PVDF or nitrocellulose membrane; pre-wet properly

• Detection system too sensitive: Reduce exposure time; dilute ECL reagent

Problem: Inconsistent results between experiments

Potential causes and solutions:

• Variable sample preparation: Standardize lysis protocol; use the same buffer consistently

• Antibody variability: Use the same lot number when possible; make large aliquots

• Loading control issues: Verify equal loading with multiple controls (e.g., GAPDH, β-actin)

• Transfer inconsistency: Use pre-stained markers to verify transfer; consider stain-free technology

• Sample degradation: Prepare fresh lysates; avoid repeated freeze-thaw cycles

Additional optimization tips:

• When troubleshooting, change only one variable at a time

• Document all experimental conditions meticulously

• For mitochondrial proteins like MRPS5, enriching for mitochondrial fraction can improve detection

• Consider using gradient gels (4-12%) for better resolution around the target molecular weight

• For publication-quality blots, test multiple antibody concentrations side by side

How can researchers distinguish between specific and non-specific binding when using MRPS5 antibodies?

Distinguishing between specific and non-specific binding is crucial for accurate interpretation of results when using MRPS5 antibodies. Several strategies can help researchers ensure specificity:

Validation controls:

• Genetic knockdown/knockout validation:

  • Use siRNA/shRNA knockdown or CRISPR knockout cells

  • The specific MRPS5 band should diminish or disappear proportionally to knockdown efficiency

  • Non-specific bands will typically remain unchanged

• Competing peptide blocking:

  • Pre-incubate antibody with excess immunizing peptide/recombinant protein

  • Specific signals should be blocked while non-specific signals persist

  • Particularly useful for validating novel antibodies or applications

• Multiple antibodies approach:

  • Use different antibodies targeting distinct epitopes of MRPS5

  • Specific signal should be detected by multiple antibodies at the same molecular weight

  • Different binding patterns suggest potential non-specific interactions

Technical approaches:

• Antibody titration:

  • Test a range of antibody dilutions (1:500 to 1:3000)

  • Specific signals typically remain visible at higher dilutions while non-specific binding diminishes

  • Create a signal-to-noise ratio curve to determine optimal concentration

• Stringent washing conditions:

  • Increase washing stringency (more TBS-T washes, higher Tween-20 concentration)

  • Specific binding is typically more resistant to stringent washing

  • Optimize washing without compromising true signal

• Subcellular fractionation:

  • MRPS5 should be enriched in mitochondrial fractions

  • Compare whole cell lysate with mitochondrial fraction

  • Specific MRPS5 bands should be enriched in mitochondrial preparations

Analytical considerations:

• Molecular weight analysis:

  • MRPS5 has a calculated molecular weight of 48 kDa but is typically observed at 38-42 kDa

  • Bands at significantly different molecular weights should be scrutinized carefully

  • Consider whether multiple bands represent isoforms, processing variants, or non-specific binding

• Relative abundance assessment:

  • MRPS5 expression varies by tissue/cell type but follows predictable patterns

  • Unusually high expression in unexpected tissues may indicate non-specific binding

  • Compare with published expression data or RNA-seq databases

• Biological validation:

  • Verify that changes in MRPS5 levels correlate with expected biological responses

  • For example, in ccRCC, MRPS5 levels correlate with specific clinical parameters

  • Disconnect between antibody signals and expected biology suggests potential specificity issues

By implementing these approaches systematically, researchers can confidently distinguish between specific MRPS5 detection and non-specific binding artifacts.

How can MRPS5 be targeted for potential therapeutic applications in cardiac disease?

The emerging role of MRPS5 in cardiac function and regeneration presents promising therapeutic opportunities for cardiac diseases:

Mechanistic basis for therapeutic targeting:

• Partial inhibition approach:

  • Complete loss of MRPS5 is embryonically lethal, but heterozygous deletion (Mrps5+/-) promotes cardiomyocyte proliferation and cardiac regeneration

  • This suggests that partial inhibition could be therapeutically beneficial

• ATF4-mediated proliferation pathway:

  • MRPS5 reduction activates ATF4 (activating transcription factor 4)

  • ATF4 regulates Knl1 (kinetochore scaffold 1)

  • This increases cytokinesis during cardiomyocyte proliferation

  • The pathway represents multiple potential intervention points

• Mitonuclear communication:

  • MRPS5 regulates Klf15 expression through c-myc and L-phenylalanine

  • Targeting this axis could rebalance cardiac metabolism in heart failure

Potential therapeutic strategies:

• Small molecule MRPS5 modulators:

  • Partial inhibitors could mimic the beneficial effects of heterozygous deletion

  • Screening libraries of FDA-approved drugs may identify compounds that modulate MRPS5 function

  • Targeted drug delivery to the heart would be essential to minimize off-target effects

• Antibiotic repurposing:

  • Certain antibiotics (e.g., doxycycline) inhibit mitochondrial translation

  • These have been shown to activate similar mechanisms as MRPS5 reduction

  • Low-dose, pulsed treatment regimens could potentially stimulate regeneration without significant antimicrobial effects

• Gene therapy approaches:

  • AAV-delivered RNAi targeting MRPS5 could achieve partial knockdown

  • CRISPR-based transcriptional repression (CRISPRi) could provide tunable reduction

  • Cardiac-specific promoters would ensure tissue-specific effects

• Downstream target modulation:

  • ATF4 activators or Klf15 modulators might bypass the need for direct MRPS5 targeting

  • This approach could potentially reduce off-target effects on mitochondrial function

Therapeutic monitoring considerations:

• Biomarkers for response:

  • Changes in mitochondrial function parameters (ATP levels, ROS production)

  • Cardiomyocyte proliferation markers (Ki67, EdU incorporation)

  • Cardiac function measurements (echocardiography)

• Safety monitoring:

  • Mitochondrial translation accuracy (potential mistranslation effects)

  • Tissue-specific effects beyond the heart

  • Long-term consequences on mitochondrial health

These therapeutic strategies require careful development to balance beneficial effects on cardiac regeneration against potential disruption of mitochondrial function in the heart and other tissues.

What is the relationship between MRPS5 and cancer progression mechanisms?

The relationship between MRPS5 and cancer appears to be complex and context-dependent, with contrasting roles across different cancer types:

Tumor suppressor role in renal cell carcinoma:

In clear cell renal cell carcinoma (ccRCC), multiple lines of evidence support a tumor suppressor role:

Potential oncogenic role in other cancers:

Contrasting observations have been made in other cancer types:

• Bladder cancer:

  • MRPS5 expression is higher in tumoral tissues than in peritumoral tissues

  • MRPS5 knockdown inhibits cell proliferation

  • Cell cycle arrest occurs in the S phase following MRPS5 inhibition

  • Cancer stem cell related transcription factors (c-Myc, Sox2, Oct4, Nanog) decrease after MRPS5 inhibition

  • Tumorigenicity in nude mice is reduced following MRPS5 knockdown

• Papillary thyroid carcinoma:

  • MRPS5 shows low expression in tumoral tissues but high expression in peritumoral tissues

  • Low expression correlates with increased risk of cervical lymph node invasion

Proposed mechanisms linking MRPS5 to cancer:

• Mitochondrial translation effects:

  • Cancer cells often rely on altered metabolic profiles

  • MRPS5-mediated changes in mitochondrial protein synthesis could affect energy metabolism

  • This may influence cancer cell growth and adaptation differently across cancer types

• Mitonuclear communication:

  • MRPS5 influences nuclear gene expression through retrograde signaling

  • In cardiac cells, this occurs through the c-myc/Klf15 axis

  • Similar pathways may be active in cancer cells, affecting proliferation and differentiation

• Stress response modulation:

  • Changes in MRPS5 can trigger mitochondrial stress responses

  • This includes ATF4 activation, which has known roles in cancer progression

  • These stress responses may be pro- or anti-tumorigenic depending on cellular context

The tissue-specific effects of MRPS5 in cancer highlight the importance of context in mitochondrial biology and suggest that therapeutic approaches targeting MRPS5 would need to be carefully tailored to specific cancer types and molecular profiles.

How might MRPS5 function interact with aging and longevity pathways?

MRPS5 has intriguing connections to aging and longevity pathways through its central role in mitochondrial translation and cellular metabolism:

Established connections to longevity:

• C. elegans studies:

  • MRPS5 has been identified as a longevity-related protein

  • It regulates lifespan through mitochondrial energy metabolism disorders

  • This establishes a direct link between mitochondrial translation and organismal aging

• Mitochondrial translation accuracy:

  • MRPS5 mutations (e.g., V336Y) cause mitoribosomal mistranslation

  • This leads to reduced ATP levels and increased reactive oxygen species

  • Both factors are hallmarks of aging across species

• Stress response pathways:

  • MRPS5 modulation activates mitochondrial stress responses

  • These include ATF4 upregulation, which influences cellular stress resistance

  • Similar stress response pathways are implicated in longevity across multiple model organisms

Potential mechanisms linking MRPS5 to aging:

Translational implications:

• Pharmacological opportunities:

  • Antibiotics targeting mitochondrial ribosomes (e.g., doxycycline) can mimic aspects of MRPS5 reduction

  • Low-dose antibiotic regimens might engage longevity pathways through controlled mitochondrial stress

  • This presents opportunities for drug repurposing in aging research

• Biomarker potential:

  • MRPS5 function or downstream responses might serve as biomarkers of aging

  • These could include measures of mitochondrial translation accuracy or specific stress responses

• Tissue-specific considerations:

  • MRPS5 effects may vary by tissue, as evidenced by its contrasting roles in different cancer types

  • Age-related interventions targeting MRPS5 pathways would need to account for these tissue-specific effects

The intersection of MRPS5 function with aging pathways represents a promising area for future research, particularly in understanding how mitochondrial translation quality control influences cellular and organismal aging.

What criteria should be used when selecting an MRPS5 antibody for specific applications?

Selecting the appropriate MRPS5 antibody requires careful consideration of multiple factors to ensure optimal performance for specific applications:

Application-specific considerations:

• Western blot (WB):

  • Validated antibodies with demonstrated specificity at the correct molecular weight (38-42 kDa observed vs. 48 kDa predicted)

  • Recommended dilutions typically range from 1:500-1:3000

  • Polyclonal antibodies often provide stronger signals for WB applications

• Immunohistochemistry (IHC):

  • Look for antibodies specifically validated for IHC/FFPE tissues

  • Check recommended antigen retrieval methods (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Typical dilutions range from 1:50-1:500

• ELISA:

  • Higher dilutions are typically used (1:2000-1:10000)

  • Verify if the antibody has been validated against recombinant MRPS5 protein

• Immunoprecipitation (IP):

  • Fewer antibodies are validated for this application

  • Look for specific IP protocols provided by the manufacturer

Technical specifications to evaluate:

• Epitope information:

  • Antibodies targeting different regions may perform differently

  • N-terminal vs. C-terminal antibodies may detect different isoforms

  • Verify that the immunogen covers functionally important domains

• Host species and clonality:

  • Rabbit polyclonal antibodies are common for MRPS5

  • Consider potential cross-reactivity with other sample components

  • For co-staining experiments, select antibodies raised in different host species

• Species reactivity:

  • Most commercial MRPS5 antibodies are validated for human samples

  • For animal models, verify cross-reactivity or select species-specific antibodies

  • Check sequence homology between species for non-validated applications

• Purification method:

  • Antigen-affinity purified antibodies typically offer higher specificity

  • Protein A/G purified antibodies may be suitable for many applications

  • Affinity purification against the immunizing protein offers higher specificity

Validation evidence to assess:

• Publication record:

  • Antibodies used in peer-reviewed publications offer greater confidence

  • Check if the antibody has been used in similar experimental contexts

• Validation data:

  • Western blot images showing a clean band at expected molecular weight

  • IHC images demonstrating appropriate subcellular localization (cytoplasmic/mitochondrial)

  • Knockdown/knockout validation data providing definitive specificity evidence

• Lot-to-lot consistency:

  • Manufacturer's quality control data

  • Certificate of analysis for each lot

  • Some manufacturers offer guaranteed lot-to-lot consistency

Additional practical considerations:

• Sample compatibility:

  • MRPS5 expression varies across cell types and tissues

  • Verify antibody performance in your specific sample type

  • Consider sensitivity requirements based on expected expression levels

• Experimental controls:

  • Availability of positive control lysates (HeLa, HEK-293, HepG2, Jurkat)

  • Possibility to generate negative controls (knockdown/knockout)

• Cost and quantity:

  • Balance between cost and quantity needed for planned experiments

  • Consider long-term needs for experimental reproducibility