MRPL3 Antibody

What is MRPL3 and why is it significant in molecular research?

MRPL3 (Mitochondrial Ribosomal Protein L3) functions as a critical component of the mitochondrial ribosome, specifically within the large 39S subunit. It belongs to the ribosomal protein L3P family and plays an essential role in mitochondrial protein biosynthesis and ribosomal structure maintenance .

The significance of MRPL3 extends beyond basic mitochondrial function, as recent research has identified its involvement in various pathological conditions. Notably, MRPL3 has been linked to:

• Combined oxidative phosphorylation deficiency 9

• Potential biomarker for hepatocellular carcinoma (HCC)

• Role in tumor growth and metastasis mechanisms

As a 348 amino acid protein localized in the mitochondrion, MRPL3 represents an important target for studying mitochondrial translation and related cellular processes .

What are the common applications of MRPL3 antibodies in research?

MRPL3 antibodies are employed across multiple experimental techniques, with the following applications being most prevalent:

Research indicates successful detection of MRPL3 in multiple human cell lines including A431, HeLa, HepG2, and in tissues such as human cervical cancer tissue and liver cancer tissue . Multiple species reactivity has been documented, with most antibodies showing high reactivity with human samples and variable cross-reactivity with mouse, rat, cow, rabbit, dog, and guinea pig specimens .

How should researchers select the appropriate MRPL3 antibody for their experiments?

Selection of an appropriate MRPL3 antibody should be guided by several critical factors:

• Target epitope region: Consider whether you need an antibody targeting the N-terminal, C-terminal, or specific amino acid regions. Different epitope-targeting antibodies exist for MRPL3:

  • N-Term antibodies (e.g., ABIN2789211)

  • C-Term antibodies

  • Specific amino acid region antibodies (AA 32-81, AA 107-156, AA 201-348, etc.)

• Host species and clonality: Most available MRPL3 antibodies are rabbit polyclonal antibodies, though some mouse polyclonal options exist . This is particularly important when planning multi-labeling experiments to avoid cross-reactivity.

• Validated applications: Ensure the antibody has been validated for your specific application. For example, antibody ABIN2789211 has been validated specifically for Western Blot , while others have been validated for multiple applications .

• Predicted reactivity across species: Consider the species compatibility based on sequence homology:

  • Cow: 93%

  • Dog: 86%

  • Guinea Pig: 93%

  • Human: 100%

  • Mouse: 79%

  • Rabbit: 100%

  • Rat: 100%

• Conjugation status: Determine whether unconjugated or conjugated (HRP, FITC, Biotin) antibodies are required for your specific application .

How can MRPL3 antibodies be optimized for detecting protein expression in hepatocellular carcinoma (HCC) samples?

Recent research has identified MRPL3 as a potential biomarker for HCC, with significant implications for diagnosis and treatment . For optimal detection in HCC samples, researchers should consider the following protocol modifications:

• Sample preparation optimization:

  • For tissue samples: Use fresh frozen or FFPE HCC tissues with appropriate controls (adjacent non-tumor tissues)

  • For cell lines: The HCC-LM3 and Hep3B cell lines have been successfully used in MRPL3 knockdown studies

• Antigen retrieval protocol refinement:

  • For IHC: Use TE buffer at pH 9.0 for optimal epitope exposure in liver tissues

  • Extended retrieval times (15-20 minutes) may improve signal in highly fibrotic HCC samples

• Antibody dilution optimization:

  • Western blot: Begin with 1:1000 dilution and titrate as needed

  • IHC: Start with 1:50 dilution for HCC tissues, which may require stronger signal detection

• Signal detection considerations:

  • MRPL3 overexpression has been documented in HCC tissues compared to adjacent normal tissues

  • Expected expression level differences: Based on experimental data, MRPL3 shows notably higher expression in all five tested HCC cell lines (Huh7, Hep3B, HepG2, HCC-LM3, Li-7) compared to the hepatic normal cell line THLE2

• Validation approach:

  • Inclusion of positive controls: HepG2 cell lysates show reliable MRPL3 expression

  • Negative controls: Use MRPL3 knockdown samples (via shRNA) as technical validation

  • For clinical samples, correlation with patient data is recommended to align with the LMRG prognostic model findings

What methodological approaches should be employed when using MRPL3 antibodies for studying mitochondrial dysfunction?

MRPL3's role in mitochondrial protein synthesis makes it valuable for investigating mitochondrial dysfunction. The following methodological approaches are recommended:

• Subcellular fractionation protocol:

  • Isolate intact mitochondria using differential centrifugation

  • Verify mitochondrial fraction purity using markers such as TOMM20 (outer membrane) and COX4 (inner membrane)

  • When analyzing MRPL3 in mitochondrial fractions, a dilution of 1:500 for Western blotting is typically sufficient

• Co-immunoprecipitation strategy:

  • Use anti-MRPL3 antibodies (preferably affinity-purified) conjugated to protein A/G beads

  • Include appropriate controls (IgG from the same species)

  • Analysis should focus on mitochondrial ribosome assembly partners

• Dual immunofluorescence approach:

  • Co-stain with mitochondrial markers (MitoTracker or anti-COX4)

  • Recommended dilution for MRPL3 antibody in IF applications: 1:100-1:200

  • Confocal microscopy with Z-stack acquisition is recommended for accurate colocalization analysis

• Mitochondrial stress induction and assessment:

  • Subject cells to mitochondrial stressors (CCCP, rotenone, antimycin A)

  • Monitor MRPL3 levels via Western blot during stress response

  • Correlate findings with mitochondrial functional parameters (membrane potential, ROS production)

• MRPL3 knockdown effect analysis:

  • Implement shRNA or siRNA approaches targeting MRPL3

  • Assess impact on mitochondrial translation using metabolic labeling

  • Evaluate effects on OXPHOS complex assembly and function

How should researchers interpret unexpected molecular weight variations when detecting MRPL3 using antibodies?

Researchers occasionally encounter unexpected molecular weight variations when detecting MRPL3. The calculated molecular weight of MRPL3 is approximately 39 kDa, but observed molecular weights typically range from 35-39 kDa . To properly interpret these variations:

• Common causes of molecular weight variations:

  • Post-translational modifications: MRPL3 may undergo phosphorylation or other modifications

  • Splice variants: Alternative splicing can generate variable protein products

  • Proteolytic processing: Mitochondrial import can involve cleavage of targeting sequences

• Validation approaches:

  • Use multiple antibodies targeting different epitopes of MRPL3

  • Implement MRPL3 knockdown controls to confirm specificity

  • Perform mass spectrometry analysis of immunoprecipitated protein

• Technical considerations:

  • Gel percentage effect: Higher percentage gels (12-15%) provide better resolution for MRPL3

  • Sample preparation impact: Denaturing conditions may affect observed molecular weight

  • Loading control selection: Use mitochondrial proteins like VDAC or COX4 rather than total cellular proteins like GAPDH when possible

• Data interpretation guidelines:

  • Document the exact molecular weight observed in your experimental system

  • Compare with literature values while considering experimental variables

  • Consider cell type-specific variations in post-translational modifications

• Troubleshooting unexpected bands:

  • For multiple bands: Assess if they represent different isoforms or degradation products

  • For higher molecular weight bands: Consider potential dimers or protein complexes

  • For lower molecular weight bands: Evaluate potential proteolytic cleavage or degradation

How can MRPL3 antibodies be utilized to investigate the role of MRPL3 in cancer progression and metastasis?

Recent research has implicated MRPL3 in cancer progression, particularly in hepatocellular carcinoma . To investigate its role:

• Tissue microarray analysis protocol:

  • Perform IHC on cancer tissue microarrays containing primary tumors and metastatic samples

  • Compare MRPL3 expression between normal, primary tumor, and metastatic tissues

  • Recommended antibody dilution: 1:50-1:100 for IHC

  • Quantify expression using digital pathology tools

• Correlation with EMT markers:

  • Research indicates potential links between MRPL3 and epithelial-mesenchymal transition

  • Co-stain or analyze sequential sections for MRPL3 alongside E-cadherin and vimentin

  • Western blot analysis shows that MRPL3 knockdown increases E-cadherin expression while decreasing vimentin expression

• Functional studies with knockdown approaches:

  • Implement shRNA or siRNA strategies to downregulate MRPL3 expression

  • Assess effects on cell proliferation using CCK-8 assay

  • Evaluate migration using wound healing assays

  • Quantify invasion using Transwell assays

  • Data from MRPL3 knockdown experiments showed:

    • Significant reduction in cell viability after 72 hours (p<0.01)

    • Increased apoptosis, particularly in late-stage

    • Inhibited migration and invasion capabilities

• Clinical sample correlation:

  • Analyze MRPL3 expression in relation to patient survival data

  • Consider incorporating MRPL3 into the LMRG prognostic model

  • The AUC value for MRPL3 in predicting HCC prognosis was reported as 0.786

• Mechanistic pathway analysis:

  • Focus on PI3K/AKT/mTOR signaling based on related mitochondrial ribosomal protein studies

  • Analyze apoptotic pathway proteins (cleaved-caspase3, cleaved-caspase9, Bcl-2)

What are the optimal protocols for using MRPL3 antibodies in single-cell analysis of tumor immune microenvironment?

Single-cell analysis has revealed MRPL3 expression patterns across various immune cell populations in the tumor microenvironment. For optimal protocols:

• Sample preparation for single-cell analysis:

  • Fresh tissue dissociation: Enzymatic digestion with collagenase/DNase

  • Cell sorting: Use CD45+ enrichment for immune cell populations

  • Fixation: Use 4% PFA for 10 minutes at room temperature

• Antibody panel design:

  • Include MRPL3 antibody (1:100 dilution) with immune cell markers

  • Consider key immune cell populations where MRPL3 is predominantly expressed:

    • Dendritic cells (DCs)

    • Innate lymphoid cells (ILCs)

    • Plasma cells

    • Proliferating T cells (Tprolif)

• Flow cytometry optimization:

  • Permeabilization: Use 0.1% Triton X-100 for intracellular staining

  • Blocking: 5% BSA for 30 minutes

  • Secondary antibody selection: Anti-rabbit IgG conjugated with bright fluorophores

• Single-cell sequencing integration:

  • Correlate protein-level data with transcriptomic profiles

  • Focus on immune cell populations with high MRPL3 expression

  • Analyze in context of immunotherapy response markers

• Data analysis approach:

  • Dimensionality reduction: Use UMAP or t-SNE for visualization

  • Clustering: Identify cell populations with differential MRPL3 expression

  • Trajectory analysis: Track MRPL3 changes across immune cell differentiation states

  • Single-cell data from GSE140228 showed that MRPL3 expression was predominantly aggregated in DC, ILC, Plasma, and Tprolif cells

How should researchers address potential cross-reactivity when using MRPL3 antibodies in complex tumor samples?

Cross-reactivity can complicate interpretation of MRPL3 staining in heterogeneous tumor samples. To address this challenge:

• Antibody validation workflow:

  • Test on positive control samples with known MRPL3 expression (HepG2, HeLa cells)

  • Validate using negative controls (MRPL3 knockdown samples)

  • Peptide competition assays to confirm specificity

• Technical controls:

  • Include isotype controls matched to the primary antibody

  • Use secondary-only controls to assess non-specific binding

  • Consider absorption controls with immunizing peptide

• Multi-antibody approach:

  • Use antibodies targeting different epitopes of MRPL3

  • Compare N-terminal vs. C-terminal targeting antibodies

  • Assess consistency of staining patterns between different antibodies

• Cross-species validation:

  • Leverage predicted reactivity data for different species:

    • Human: 100%

    • Mouse: 79%

    • Rat: 100%

    • Cow: 93%

    • Dog: 86%

    • Guinea Pig: 93%

    • Rabbit: 100%

  • Particularly important for xenograft models with mixed human/mouse tissues

• Confounding factors assessment:

  • Tissue fixation effects: Different fixatives may affect epitope accessibility

  • Tumor heterogeneity: Account for variable expression across tumor regions

  • Necrotic areas: Evaluate potential non-specific binding in necrotic regions

What are the most effective strategies for troubleshooting weak or absent MRPL3 signal in Western blot applications?

When encountering weak or absent MRPL3 signal in Western blots, consider the following troubleshooting strategies:

• Sample preparation optimization:

  • Ensure complete cell lysis using RIPA buffer with protease inhibitors

  • For mitochondrial proteins like MRPL3, consider mitochondrial enrichment protocols

  • Avoid excessive freeze-thaw cycles of samples

• Protein loading considerations:

  • Increase loading amount (30-50 μg of whole cell lysate is typically recommended)

  • For cell lines with lower MRPL3 expression, consider loading up to 50-60 μg

• Transfer efficiency improvements:

  • For MRPL3 (35-39 kDa), use 0.2 μm PVDF membranes rather than nitrocellulose

  • Optimize transfer conditions: 100V for 60-90 minutes in cold transfer buffer

  • Add 0.1% SDS to transfer buffer to improve elution of proteins from gel

• Antibody incubation optimization:

  • Increase primary antibody concentration (try 1:500 if using 1:1000)

  • Extend primary antibody incubation to overnight at 4°C

  • Use 5% BSA instead of milk for blocking and antibody dilution

• Detection sensitivity enhancement:

  • Use high-sensitivity ECL substrates for chemiluminescence detection

  • Increase exposure time incrementally

  • Consider using signal enhancers like SuperSignal™ Western Blot Enhancer

• Controls to include:

  • Positive control: HeLa or HepG2 cell lysates show reliable MRPL3 expression

  • Loading control: Consider mitochondrial markers (VDAC, COX4) alongside general loading controls (GAPDH, β-actin)

How can researchers optimize MRPL3 antibody-based immunoprecipitation protocols for protein interaction studies?

To optimize immunoprecipitation (IP) protocols for MRPL3 interaction studies:

• Lysis buffer selection and optimization:

  • Use gentle lysis buffers to preserve protein interactions:

    • Standard IP buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate

    • For mitochondrial proteins: Add 1 mM EDTA and 2 mM MgCl₂

  • Include protease and phosphatase inhibitor cocktails

• Pre-clearing strategy:

  • Pre-clear lysates with Protein A/G beads (40 μl of 50% slurry per 1 mg protein)

  • Incubate for 1 hour at 4°C with gentle rotation

  • Remove beads by centrifugation at 2,500g for 5 minutes

• Antibody binding optimization:

  • Use affinity-purified antibodies when available

  • Typical antibody amount: 2-5 μg per 500 μg of lysate

  • Incubation time: Overnight at 4°C with gentle rotation

• Washing procedure refinement:

  • Perform 4-5 washes with decreasing salt concentrations

  • First wash: Lysis buffer with 300 mM NaCl

  • Final wash: 50 mM Tris-HCl pH 7.4

  • Keep samples cold throughout the procedure

• Elution method selection:

  • Gentle elution: 0.1 M glycine pH 3.0, neutralize immediately with 1M Tris pH 8.0

  • Denaturing elution: 1X SDS sample buffer at 95°C for 5 minutes

  • Native elution: Competitive displacement with immunizing peptide

• Controls and validation:

  • Input control: 5-10% of lysate used for IP

  • Negative control: Non-specific IgG from same species as MRPL3 antibody

  • Reciprocal IP: Confirm interactions by IP with antibodies against suspected interaction partners

What considerations should be taken into account when using MRPL3 antibodies for quantitative analysis across different experimental conditions?

For reliable quantitative analysis of MRPL3 across different experimental conditions:

• Standardization of sample processing:

  • Use consistent cell numbers/tissue amounts across samples

  • Process all samples simultaneously using identical protocols

  • Implement standardized protein quantification methods (BCA or Bradford)

• Reference standard implementation:

  • Create standard curves using recombinant MRPL3 protein

  • Include internal reference samples across multiple experiments

  • Consider spike-in controls for normalization

• Appropriate normalization strategy:

  • For Western blots: Normalize to housekeeping proteins appropriate for the context

  • For mitochondrial studies: Normalize to mitochondrial mass markers (VDAC, TOM20)

  • For cell-type specific studies: Consider cell-type specific markers

• Technical replicate design:

  • Minimum of three technical replicates per condition

  • Account for gel-to-gel variation in Western blot experiments

  • Use randomization in sample loading order

• Statistical approach considerations:

  • Test for normal distribution before selecting statistical tests

  • Apply appropriate multiple testing corrections

  • Consider power analysis to determine sample size requirements

  • Report effect sizes alongside p-values

• Method-specific considerations:

  • Western blot: Ensure linear dynamic range of detection

  • ELISA: Validate antibody specificity with recombinant standards

  • IHC/IF: Implement digital image analysis with defined thresholds

  • Flow cytometry: Use appropriate compensation and gating strategies

How might MRPL3 antibodies be utilized in studying the relationship between mitochondrial dysfunction and cancer metabolism?

MRPL3's role in mitochondrial translation positions it as a valuable target for investigating connections between mitochondrial function and cancer metabolism:

• Metabolic profiling correlation:

  • Analyze MRPL3 expression in relation to metabolic phenotypes

  • Correlate with glycolytic vs. oxidative phosphorylation dependence

  • Investigate lactate production and consumption patterns

• Mitochondrial translation assessment:

  • Use MRPL3 antibodies to assess mitochondrial ribosome integrity

  • Correlate with translation of key electron transport chain components

  • Evaluate impact on mitochondrial-encoded vs. nuclear-encoded OXPHOS subunits

• Tumor microenvironment studies:

  • Investigate MRPL3 expression in hypoxic vs. normoxic tumor regions

  • Correlate with HIF-1α expression and target genes

  • Recent research suggests connections between MRPL3 and metabolic adaptation in tumor progression

• Therapeutic resistance mechanisms:

  • Assess MRPL3 expression changes in response to metabolic inhibitors

  • Study potential role in resistance to drugs targeting mitochondrial function

  • Investigate synergistic approaches combining MRPL3 targeting with metabolic inhibitors

• Integration with multi-omics data:

  • Correlate protein-level MRPL3 data with:

    • Metabolomics profiles (particularly TCA cycle intermediates)

    • Transcriptomic data on metabolic enzymes

    • Proteomic data on mitochondrial complexes

  • The LMRG model incorporating MRPL3 shows connections to metabolic reprogramming pathways

What are the implications of recent MRPL3 findings for developing novel therapeutic approaches?

Recent findings on MRPL3's role in cancer progression suggest several therapeutic implications:

• Targeting strategies based on molecular findings:

  • siRNA/shRNA approaches: Knockdown studies have demonstrated anti-tumor effects

  • Small molecule inhibitors: Target MRPL3-dependent mitochondrial translation

  • Peptide mimetics: Disrupt MRPL3 interactions with ribosomal assembly factors

• Combination therapy opportunities:

  • MRPL3 targeting with chemotherapeutic agents

  • Potential synergy with drugs identified in sensitivity studies:

    • ML323 (USP1 inhibitor)

    • Selumetinib

    • AZD2014

    • Doramapimod

    • SB505124

• Biomarker development applications:

  • MRPL3 as prognostic biomarker in HCC and potentially other cancers

  • Integration into multi-gene panels like the LMRG model

  • Predictive biomarker for response to metabolic-targeting therapies

• Immunotherapeutic connections:

  • MRPL3's correlation with M2 macrophage infiltration suggests immunomodulatory potential

  • Targeting MRPL3 may help rebalance the M1/M2 ratio in the tumor microenvironment

  • Potential to enhance CD4+ T memory cell generation

• Translational research directions:

  • Develop IHC-based clinical assays for MRPL3 assessment in tumors

  • Establish cutoffs for high vs. low expression with prognostic significance

  • Compare with traditional biomarkers (e.g., AFP for HCC) to determine additive value

  • MRPL3 demonstrated superior predictive power compared to traditional HCC biomarkers like AFP, DCP, and GPC3

How can MRPL3 antibodies contribute to understanding the intersection of mitochondrial ribosome dysfunction and neurodegenerative diseases?

While much MRPL3 research has focused on cancer, there are significant implications for neurodegenerative disease research:

• Neurodegenerative disease models assessment:

  • Analyze MRPL3 expression in brain tissues from neurodegenerative disease models

  • Compare expression across different brain regions with varying vulnerability

  • Correlate with mitochondrial morphology and distribution in neurons

• Mitochondrial translation defects characterization:

  • Use MRPL3 antibodies to assess mitochondrial ribosome integrity in affected neurons

  • Correlate with expression of mitochondrial-encoded respiratory chain components

  • Investigate potential compensatory mechanisms in response to translation defects

• Protein aggregation interactions:

  • Explore potential interactions between MRPL3 and disease-associated proteins (Aβ, tau, α-synuclein)

  • Assess impact of protein aggregates on mitochondrial translation efficiency

  • Investigate sequestration of mitochondrial ribosomal proteins in protein aggregates

• Oxidative stress response analysis:

  • Analyze MRPL3 expression changes under oxidative stress conditions

  • Correlate with markers of mitochondrial oxidative damage

  • Assess potential protective or detrimental roles in stress response

• Therapeutic target validation:

  • Evaluate MRPL3 modulation as a potential therapeutic approach

  • Test compounds that stabilize mitochondrial ribosomes

  • Investigate molecules that enhance mitochondrial translation fidelity under stress conditions