RPS3A Antibody

What is RPS3A and why is it important in research?

RPS3A (Ribosomal Protein S3A) is a component of the 40S ribosomal small subunit involved in protein synthesis. Beyond its canonical role in translation, RPS3A has multiple non-ribosomal functions that make it a significant research target. It belongs to the S3AE family of ribosomal proteins and is also known as Fte-1, FTE1, MFTL, and v-fos transformation effector protein .

RPS3A's importance in research stems from:

• Its dual localization in both cytoplasm and nucleus

• Role in cellular transformation (originally identified as v-fos transformation effector)

• Correlation with tumor development and progression

• Involvement in immune response modulation

• Recently discovered functions in mitochondrial processes and adipocyte differentiation

Research has shown that RPS3A expression levels correlate with clinical outcomes in certain cancers, making it a potential biomarker and therapeutic target .

What experimental applications can an RPS3A antibody be used for?

RPS3A antibodies have been validated for multiple experimental techniques:

Multiple independent studies have confirmed these applications across human, mouse, rat, and other species samples .

How do I select the appropriate RPS3A antibody for my experiment?

Selection of the optimal RPS3A antibody depends on several key factors:

• Target epitope: Different antibodies target different regions (N-terminal, C-terminal, or internal epitopes). For example, ABIN2786535 targets the N-terminal region of RPS3A , while other antibodies target C-terminal regions . The epitope choice affects:

  • Cross-reactivity with species of interest

  • Accessibility in folded/denatured protein

  • Potential interference with protein-protein interactions

• Species reactivity: Based on sequence identity, most RPS3A antibodies show reactivity with human, mouse, and rat samples. Some have broader predicted reactivity including cow, dog, guinea pig, horse, zebrafish, and other species .

• Clonality:

  • Polyclonal antibodies offer broader epitope recognition but batch-to-batch variation

  • Monoclonal antibodies provide consistency but may be more sensitive to epitope modifications

• Validation data: Review Western blot images, IHC staining patterns, and knockout validation data provided by manufacturers to ensure specificity .

What are the optimal protocols for using RPS3A antibodies in Western blotting?

For optimal Western blot results with RPS3A antibodies:

Sample Preparation:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Sonicate briefly to shear DNA and reduce viscosity

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Determine protein concentration by BCA or Bradford assay

• Denature samples at 95°C for 5 minutes in Laemmli buffer with DTT or β-mercaptoethanol

Gel Electrophoresis and Transfer:

• Load 20-30 μg of protein per lane on 10-12% SDS-PAGE gels

• Use wet transfer to PVDF membrane (0.45 μm) at 100V for 60-90 minutes

Antibody Incubation:

• Block in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary RPS3A antibody (1:1000-1:5000 dilution) overnight at 4°C

• Wash 3 × 10 minutes with TBST

• Incubate with secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

• Wash 3 × 10 minutes with TBST

Expected Results:

• RPS3A typically appears at approximately 30-35 kDa

• Validation controls should include known positive cell lines (HeLa, A431, HepG2)

Multiple published studies have confirmed the effectiveness of these protocols .

How should I optimize immunohistochemistry protocols for RPS3A detection in tissue samples?

For optimal IHC results with RPS3A antibodies in FFPE tissue sections:

Antigen Retrieval (Critical Step):

• Heat-induced epitope retrieval is recommended:

  • Citrate buffer (pH 6.0) works well for most RPS3A antibodies

  • Some antibodies perform better with TE buffer (pH 9.0)

  • Pressure cooker method: 20 minutes at high pressure

Staining Protocol:

• Deparaffinize and rehydrate sections through xylene and graded alcohols

• Perform antigen retrieval as above

• Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Block non-specific binding with 5% normal serum for 1 hour

• Incubate with primary RPS3A antibody (1:400-1:1600 dilution) overnight at 4°C

• Apply appropriate detection system (HRP/DAB recommended)

• Counterstain with hematoxylin, dehydrate, and mount

Tissue-Specific Considerations:

• Human colon tissue serves as a reliable positive control

• Nuclear and cytoplasmic staining patterns are expected

• Perform parallel staining with isotype control antibody to assess background

Published studies have successfully used RPS3A antibodies for IHC in liver, colon, and adipose tissue samples .

What considerations are important when using RPS3A antibodies for co-localization studies?

When performing co-localization studies with RPS3A:

Experimental Design:

• Antibody compatibility: Ensure primary antibodies are from different host species to avoid cross-reactivity of secondary antibodies

• Fluorophore selection: Choose fluorophores with minimal spectral overlap (e.g., Alexa 488 for RPS3A and Alexa 594 for the co-target)

• Controls: Include single-antibody controls to assess bleed-through

Promising Co-localization Targets:

• Mitochondrial markers: Research has demonstrated RPS3A localization to mitochondria, especially in brown adipocytes. MitoTracker or antibodies against mitochondrial proteins (e.g., TOM20) can be used

• Ribosomal markers: Co-staining with other small ribosomal subunit proteins

• Nucleolar markers: For examining nuclear RPS3A localization

Analysis Methods:

• Calculate Pearson's correlation coefficient or Manders' overlap coefficient

• Use orthogonal or 3D reconstruction views to confirm true co-localization

• Perform quantitative analysis across multiple cells/fields

Research has shown that RPS3A co-localizes with mitochondria but not with tubulin in certain cell types, suggesting specific subcellular targeting .

How does RPS3A expression correlate with immune cell infiltration in tumor microenvironments?

Recent research has revealed significant correlations between RPS3A expression and tumor immune microenvironment:

Key Findings:

• High RPS3A expression negatively correlates with immune cell infiltration in hepatocellular carcinoma (HCC)

• Single-sample gene set enrichment analysis (ssGSEA) demonstrated strong negative correlation (r = -0.42, P < 0.001) between RPS3A expression and B cell infiltration

• Neutrophils (r = -0.3, P < 0.001) and dendritic cells (r = -0.24, P < 0.001) also showed significant negative correlations with RPS3A expression

• Weak positive correlations were observed between RPS3A and NK cells (CD56 bright r = 0.28; CD56 dim r = 0.27)

Methodological Approach:

• RPS3A expression can be assessed via RNA-seq, microarray, or qRT-PCR

• Immune cell infiltration can be evaluated through:

  • Computational methods: ssGSEA, CIBERSORT, MCP-counter

  • Experimental validation: Multiplex IHC, flow cytometry

• Correlation analysis using Spearman rank correlation

Clinical Implications:

• RPS3A expression positively correlates with expression of immune checkpoint molecules

• High RPS3A expression is associated with poor prognosis in HCC patients

• RPS3A-based nomograms show superior predictive accuracy compared to traditional staging systems

What is the significance of RPS3A in mitochondrial function and how can this be studied?

Emerging research indicates RPS3A plays a significant role in mitochondrial function:

Research Findings:

• RPS3A can migrate to mitochondria to maintain brown adipocyte function

• Knockdown of RPS3A impairs mitochondrial function in mature adipocytes

• RPS3A appears to regulate brown fat-specific genes like UCP-1 and carbon metabolic enzymes

• RPS3A expression is decreased in epicardial adipose tissue from coronary artery disease patients

Experimental Approaches to Study This Function:

• Subcellular Fractionation:

  • Isolate mitochondrial fractions using differential centrifugation

  • Confirm purity using markers for mitochondria, cytosol, and other organelles

  • Analyze RPS3A presence in different fractions by Western blot

• Imaging Approaches:

  • Co-localization studies using confocal microscopy

  • Super-resolution microscopy for detailed spatial relationships

  • Live-cell imaging to track RPS3A translocation

• Functional Assays:

  • Oxygen consumption rate (OCR) measurement using Seahorse analyzer

  • Mitochondrial membrane potential assays

  • Analysis of TCA cycle metabolites and β-oxidation after RPS3A manipulation

• Gene Expression Analysis:

  • qPCR for mitochondrial genes after RPS3A knockdown/overexpression

  • RNA-seq to assess global transcriptomic changes

  • ChIP-seq to identify potential direct gene targets

Research has shown that RPS3A knockdown decreases oxygen consumption rate in brown adipocytes, suggesting direct involvement in mitochondrial function .

How can RPS3A knockdown/knockout models be effectively generated and validated?

Creating reliable RPS3A knockdown/knockout models requires careful consideration of several factors:

RNAi-Based Knockdown:

• siRNA design considerations:

  • Target sequence specificity (avoid off-target effects)

  • Efficiency of knockdown (typically 70-90% reduction)

  • Duration of effect (transient, typically 3-7 days)

• Validation methods:

  • qRT-PCR to assess mRNA reduction

  • Western blot to confirm protein depletion

  • Include scrambled siRNA controls

  • Test multiple siRNA sequences targeting different regions

CRISPR/Cas9 Knockout:

• gRNA design considerations:

  • Target early exons to ensure functional knockout

  • Account for potential alternative transcripts

  • Check for off-target sites

• Validation strategies:

  • Genomic PCR and sequencing to confirm mutations

  • Western blot to confirm complete protein absence

  • Functional assays to demonstrate phenotype

  • Rescue experiments to confirm specificity

Challenges Specific to RPS3A:

• Complete knockout may be lethal due to essential ribosomal function

• Compensatory upregulation of related proteins may occur

• Cell type-specific effects may require tissue-specific models

Successful Applications:
Studies have effectively used RPS3A knockdown in:

• Brown preadipocytes (showing inhibition of adipogenic ability)

• HCC cell lines (demonstrating effects on tumor cell proliferation)

• Mature brown adipocytes (revealing mitochondrial dysfunction)

What are common problems with RPS3A antibody experiments and how can they be addressed?

Researchers frequently encounter these challenges when working with RPS3A antibodies:

For epitope-specific issues:

• N-terminal antibodies may be affected by protein processing

• C-terminal antibodies might not recognize truncated forms

• Consider using antibodies targeting different regions for verification

Successful researchers employ multiple validation approaches, including positive and negative controls, to ensure specificity .

How can I troubleshoot inconsistent RPS3A staining patterns in different tissue types?

Inconsistent RPS3A staining across tissue types can result from several factors:

Causes and Solutions:

• Fixation differences:

  • Duration and type of fixation affect epitope preservation

  • Solution: Standardize fixation protocols (e.g., 24h in 10% neutral buffered formalin)

  • For frozen sections, ensure consistent fixation in 4% paraformaldehyde

• Antigen retrieval optimization:

  • Different tissues may require different AR methods

  • Solution: Test both citrate (pH 6.0) and TE buffer (pH 9.0) for each tissue type

  • Optimize AR time (15-30 minutes) for each tissue

• Endogenous peroxidase/phosphatase activity:

  • Variable levels across tissue types

  • Solution: Extend blocking time for tissues with high activity (liver, kidney)

  • Use dual blocking approach (H₂O₂ + levamisole for phosphatase)

• Tissue-specific expression levels:

  • RPS3A expression varies naturally between tissues

  • Solution: Adjust antibody concentration for each tissue type

  • Include known positive controls (colon, liver) alongside test samples

• Background reduction:

  • For high-background tissues, add:

    • 0.3% Triton X-100 to improve antibody penetration

    • 1% BSA to reduce nonspecific binding

    • Avidin/biotin blocking for biotin-rich tissues

Research has shown that RPS3A expression patterns differ significantly between adipose tissue types and hepatocellular tissue, requiring tissue-specific optimization .

What controls should be included when using RPS3A antibodies for quantitative analysis?

Robust quantitative analysis with RPS3A antibodies requires comprehensive controls:

Essential Controls:

• Positive Controls:

  • Cell lines with known RPS3A expression (HeLa, MCF7, HepG2)

  • Tissues with consistent RPS3A expression (colon, liver)

  • Recombinant RPS3A protein (for standard curve in quantitative applications)

• Negative Controls:

  • RPS3A knockdown/knockout samples when available

  • Primary antibody omission

  • Isotype control antibody (matched concentration)

• Loading/Normalization Controls:

  • For Western blot: Housekeeping proteins (β-actin, GAPDH, α-tubulin)

  • For IHC/IF: Adjacent serial sections with standardized markers

  • For flow cytometry: Viability markers, isotype controls

• Quantification Standards:

  • Include calibration samples with known RPS3A concentrations

  • Run dilution series to ensure detection within linear range

  • Use identical image acquisition settings for all compared samples

Statistical Considerations:

• Perform experiments in at least triplicate

• Use appropriate statistical tests based on data distribution

• Account for batch effects in multi-experiment analyses

Special Considerations for RPS3A:
Since RPS3A is a ribosomal protein with high basal expression in many tissues, dynamic range and sensitivity are important factors in quantitative applications .

How do I interpret changes in RPS3A localization between cytoplasmic and nuclear compartments?

RPS3A's dynamic localization pattern provides important functional insights:

Normal Distribution Pattern:

• Predominantly cytoplasmic (associated with ribosomes)

• Nucleolar localization during ribosome biogenesis

• Occasional diffuse nuclear staining

Altered Distribution Patterns and Their Interpretation:

• Increased Nuclear Localization:

  • Often indicates cellular stress response

  • May reflect ribosome biogenesis dysregulation

  • Could suggest non-canonical functions in transcriptional regulation

  • Correlation with tumor grade in some cancers

• Enhanced Nucleolar Concentration:

  • Associated with increased ribosome synthesis

  • Common in rapidly proliferating cells

  • Observed in certain metabolically active tissues

• Mitochondrial Localization:

  • Recently identified pattern, especially in brown adipocytes

  • Suggests role in mitochondrial function

  • Can be verified by co-localization with mitochondrial markers

  • Reduced in pathological conditions like coronary artery disease

Quantification Approaches:

• Nuclear/cytoplasmic ratio measurement

• Subcellular fractionation followed by Western blotting

• High-resolution imaging with quantitative co-localization analysis

Research has shown that RPS3A localization changes can occur in response to cellular stressors and during differentiation processes, providing insight into its non-canonical functions .

What is the significance of post-translational modifications of RPS3A in experimental results?

Post-translational modifications (PTMs) of RPS3A significantly impact its function and detection:

Known RPS3A PTMs:

• Phosphorylation: Documented at T10 and S26 sites

• Acetylation: Observed at K27

• Other potential modifications: Ubiquitination, SUMOylation

Experimental Implications:

• Antibody Epitope Considerations:

  • PTMs may mask epitopes, reducing antibody binding

  • Modifications near the antibody binding site can affect detection efficiency

  • Some antibodies may preferentially recognize modified/unmodified forms

• Detection Methods for Modified RPS3A:

  • Phospho-specific antibodies for key phosphorylation sites

  • Lambda phosphatase treatment to confirm phosphorylation

  • 2D gel electrophoresis to separate modified forms

  • Mass spectrometry for comprehensive PTM mapping

• Functional Significance:

  • Phosphorylation may regulate nuclear-cytoplasmic shuttling

  • Acetylation could affect protein-protein interactions

  • PTMs may redirect RPS3A to non-ribosomal functions

  • Modified forms may have altered stability or localization

Interpretation Guidelines:

• Multiple bands near the expected molecular weight may represent different PTM states

• Shifts in band patterns following treatments may indicate PTM changes

• Consider using PTM inhibitors to clarify the identity of observed forms

Research suggests that RPS3A modifications may play key roles in regulating its non-canonical functions, particularly in stress response and disease conditions .

How should contradictory results between RPS3A protein and mRNA expression be reconciled?

Discrepancies between RPS3A protein and mRNA levels are not uncommon and require careful analysis:

Common Scenarios:

• High mRNA/Low protein:

  • Post-transcriptional regulation (miRNAs, RNA-binding proteins)

  • Enhanced protein degradation

  • Translational inhibition

• Low mRNA/High protein:

  • Increased protein stability/half-life

  • Reduced protein turnover

  • Post-transcriptional regulatory mechanisms

  • Antibody cross-reactivity issues

Resolution Approaches:

• Technical Validation:

  • Confirm antibody specificity via knockdown/knockout

  • Verify primer specificity for RPS3A mRNA detection

  • Check for potential detection of pseudogenes (RPS3A has multiple pseudogenes)

• Biological Mechanism Investigation:

  • Measure protein half-life (cycloheximide chase)

  • Assess proteasomal degradation (proteasome inhibitors)

  • Evaluate miRNA targeting (reporter assays)

  • Analyze polysome association (polysome profiling)

• Integrated Analysis:

  • Combine transcriptomics, proteomics, and ribosome profiling

  • Examine translation efficiency metrics

  • Consider tissue-specific regulatory mechanisms

Research Context:
Studies have shown that RPS3A can be regulated at multiple levels, and its expression pattern in cancer and immune contexts may involve complex regulatory mechanisms beyond simple transcriptional control .

What emerging applications of RPS3A antibodies show promise for cancer research?

Recent findings suggest several promising applications of RPS3A antibodies in cancer research:

Emerging Applications:

• Prognostic/Predictive Biomarker Development:

  • RPS3A expression correlates with poor prognosis in hepatocellular carcinoma

  • RPS3A-based nomograms outperform traditional staging systems

  • Potential marker for immunotherapy response prediction based on correlation with immune checkpoint molecules

• Therapeutic Target Validation:

  • Antibody-based validation of RPS3A as a potential therapeutic target

  • Monitoring RPS3A changes during experimental treatments

  • Analysis of RPS3A interaction with oncogenic pathways

• Tumor Microenvironment Assessment:

  • Multiplex IHC combining RPS3A with immune cell markers

  • Spatial relationship between RPS3A-expressing cells and tumor-infiltrating lymphocytes

  • Correlation with treatment response patterns

• Non-Canonical Function Exploration:

  • Investigation of RPS3A's roles beyond protein synthesis

  • Targeted analysis of RPS3A in specific subcellular compartments

  • Identification of cancer-specific RPS3A interaction networks

Methodological Advances:

• Proximity ligation assays to detect RPS3A-protein interactions in situ

• Mass cytometry (CyTOF) incorporating RPS3A detection

• Single-cell analysis of RPS3A expression heterogeneity within tumors

Research has demonstrated that RPS3A expression patterns correlate with immune infiltration and clinical outcomes, suggesting significant potential for diagnostic and therapeutic applications .

How might RPS3A antibodies contribute to understanding the relationship between metabolism and immune function?

The emerging role of RPS3A at the intersection of metabolism and immunity offers several research opportunities:

Key Research Areas:

• Adipose Tissue Immunobiology:

  • RPS3A's role in brown adipose tissue function and immunomodulation

  • Connection between RPS3A, mitochondrial activity, and immune cell infiltration

  • Potential therapeutic targeting in inflammatory conditions associated with adipose dysfunction

• Mitochondrial Function in Immune Cells:

  • RPS3A localization in immune cell mitochondria

  • Impact of RPS3A expression on immune cell metabolism and function

  • Mechanistic studies of how RPS3A regulates metabolic pathways in different immune cell subsets

• Tumor Metabolism and Immune Evasion:

  • Correlation between RPS3A, metabolic reprogramming, and immune checkpoint expression

  • Impact of RPS3A on tumor microenvironment metabolites

  • Mechanistic studies linking RPS3A's metabolic functions to immunosuppression

Experimental Approaches:

• Multi-parameter flow cytometry combining RPS3A with metabolic and immune markers

• Seahorse analysis of metabolic profiles in immune cells after RPS3A modulation

• Metabolomic analysis paired with RPS3A expression profiling

• In vivo models examining RPS3A's role in immunometabolism

Research has shown negative correlations between RPS3A expression and immune cell infiltration in tumors, while also identifying roles for RPS3A in mitochondrial function, suggesting a potential mechanistic link between these phenomena .

What new technologies and methods might enhance the specificity and applications of RPS3A antibodies?

Emerging technologies offer exciting possibilities for advancing RPS3A research:

Technological Advances:

• Next-Generation Antibody Formats:

  • Single-domain antibodies (nanobodies) for improved tissue penetration

  • Bispecific antibodies targeting RPS3A and interaction partners

  • Intrabodies for tracking RPS3A in living cells

  • Conditional antibodies activated in specific cellular compartments

• Advanced Imaging Techniques:

  • Super-resolution microscopy (STORM, PALM) for detailed localization studies

  • Lattice light-sheet microscopy for live-cell dynamics

  • Expansion microscopy for enhanced spatial resolution

  • Correlative light and electron microscopy for ultrastructural context

• Single-Cell Applications:

  • Single-cell Western blotting for heterogeneity assessment

  • Mass cytometry (CyTOF) incorporating RPS3A detection

  • Spatial transcriptomics combined with RPS3A protein mapping

  • Microfluidic approaches for single-cell proteomic analysis

• Computational Methods:

  • Machine learning algorithms for automated quantification of RPS3A localization

  • Network analysis tools to understand RPS3A interaction hubs

  • Systems biology approaches integrating multi-omics data with RPS3A function

Application Expansion:

• Antibody-based proximity labeling to identify context-specific interactors

• CRISPR screening combined with RPS3A antibody-based readouts

• Organoid and tissue chip applications for physiologically relevant models

These technological advances could significantly enhance our understanding of RPS3A's diverse functions in normal physiology and disease states .