RPS8 Antibody

What is RPS8 and what is its biological significance?

RPS8 (Ribosomal Protein S8) is an essential component of the 40S small ribosomal subunit involved in protein synthesis. It belongs to the S8E family of ribosomal proteins and is primarily located in the cytoplasm. Ribosomes consist of small 40S and large 60S subunits, together comprising 4 RNA species and approximately 80 structurally distinct proteins . Beyond its canonical role in translation, RPS8 functions as a rate-limiting factor in translational regulation and contributes to stress responses such as cold-adaptability in plants .

RPS8 has gained research interest due to its altered expression in several disease states. Increased expression has been observed in colorectal tumors and colon polyps compared to matched normal colonic mucosa, suggesting potential roles in carcinogenesis . Recent studies have also identified RPS8 as a novel biomarker specifically for alcohol-associated hepatocellular carcinoma (HCC) . This protein is encoded by a gene that is co-transcribed with small nucleolar RNA genes U38A, U38B, U39, and U40, which are located in its introns .

What types of RPS8 antibodies are commercially available?

Several types of RPS8 antibodies are available for research applications, primarily as polyclonal antibodies raised in rabbits:

Each antibody is generated using different immunogens. For example, Proteintech's antibody uses an RPS8 fusion protein (Ag12313) , while Boster's antibody utilizes a synthesized peptide derived from human RPS8 (amino acids 111-160) . These differences in immunogen may affect epitope recognition and performance in specific applications.

What applications are validated for RPS8 antibodies?

RPS8 antibodies have been validated for multiple research applications:

• Western Blot (WB):

  • Successfully detects RPS8 in various cell lines (HeLa, SGC-7901, PC-3, A375) and tissues (mouse liver, pancreas)

  • Recommended dilution for Proteintech 18228-1-AP: 1:500-1:2000

• Immunohistochemistry (IHC):

  • Used for detecting RPS8 expression in tissue sections, particularly in cancer studies

  • Successfully employed in HCC research at 1:40 dilution

• Immunofluorescence (IF)/Immunocytochemistry (ICC):

  • For subcellular localization studies

  • Validated in A375 cells with recommended dilution of 1:20-1:200 for Proteintech antibody

• ELISA:

  • Listed as an application for Proteintech 18228-1-AP

• Research Applications:

  • Biomarker studies in hepatocellular carcinoma

  • Protein interaction studies, particularly in plant research using techniques like Y2H and BiFC

The optimal application depends on your specific research question, with different antibodies showing varying performance across applications. Validation in your specific experimental system is recommended.

How should I optimize RPS8 antibody dilutions for different applications?

Optimal dilution determination is critical for achieving specific signal with minimal background. For RPS8 antibodies, follow these methodological approaches:

• Start with manufacturer recommendations:

  • Western Blot: 1:500-1:2000 (Proteintech 18228-1-AP)

  • IF/ICC: 1:20-1:200 (Proteintech 18228-1-AP)

  • IHC: 1:40 as validated in HCC research

• Perform systematic titration:

  • Prepare a dilution series spanning the recommended range (and beyond if necessary)

  • Use consistent samples with known RPS8 expression

  • Include positive controls (e.g., HeLa cells, mouse liver tissue) which have been validated

  • Include negative controls (primary antibody omission, non-target tissues)

• Evaluate signal-to-noise ratio:

  • The optimal dilution provides strong specific signal with minimal background

  • For WB: Clear band at expected molecular weight (25-28 kDa for Proteintech antibody) with minimal non-specific bands

  • For IHC/IF: Specific cellular or subcellular staining pattern with minimal background

• Consider system-specific factors:

  • Sample type may affect optimal dilution (cell lines vs. tissues, human vs. mouse)

  • Detection method sensitivity influences required antibody concentration

  • Fixation and antigen retrieval methods may necessitate dilution adjustments

Remember that "sample-dependent optimization is recommended" for all applications, and each new experimental system may require re-optimization.

What is the recommended protocol for RPS8 immunohistochemistry?

Based on published research using RPS8 antibodies for IHC in hepatocellular carcinoma studies, the following protocol has been validated:

• Tissue Preparation:

  • Fix samples in 4% paraformaldehyde for 24h at room temperature

  • Dehydrate using 98% ethyl alcohol at 40°C

  • Embed in paraffin and section at 4μm thickness

• Deparaffinization and Rehydration:

  • Deparaffinize sections with xylene

  • Rehydrate through descending alcohol series at room temperature

• Antigen Retrieval:

  • Heat-induced epitope retrieval using sodium citrate buffer at 100°C

• Blocking:

  • Block endogenous peroxidase with 3% hydrogen peroxide

  • Block non-specific binding with 5% BSA for 30 min at room temperature

• Antibody Incubation:

  • Primary antibody: Anti-RPS8 (1:40 dilution, Proteintech 18228-1-AP) for 12h at 4°C

  • Secondary antibody: HRP-conjugated secondary antibody (1:100) for 2h at room temperature

• Detection and Visualization:

  • Develop using HRP-DAB kit according to manufacturer's protocol

  • Image capture: Orthophoto light microscope (magnification, ×200)

• Scoring and Analysis:

  • Evaluation scale: 0 (0-1% positive cells), 1 (1-33%), 2 (34-66%), 3 (67-100%)

  • Image analysis using software such as Image-Pro Plus for objective quantification

This protocol has been successfully employed in studies identifying RPS8 as a biomarker for alcohol-associated HCC, demonstrating its reliability for tissue-based RPS8 detection.

How can I verify the specificity of my RPS8 antibody staining?

Confirming antibody specificity is crucial for ensuring reliable research outcomes. For RPS8 antibodies, implement these validation strategies:

• Genetic Validation:

  • RNAi knockdown: Transfect cells with RPS8-targeting siRNA and confirm reduced signal

  • Overexpression: Express RPS8 in appropriate cells and verify increased signal intensity

  • Use these genetically modified samples as positive and negative controls

• Peptide Competition Assay:

  • Pre-incubate RPS8 antibody with excess immunizing peptide

  • Compare staining with and without peptide competition

  • Specific staining should be eliminated or significantly reduced

  • For some antibodies like Boster Bio A07839, blocking peptides can be purchased

• Multiple Antibody Validation:

  • Use different RPS8 antibodies targeting distinct epitopes

  • Consistent patterns across different antibodies increase confidence in specificity

  • Compare results from different sources (e.g., Proteintech vs. Thermo Fisher)

• Technical Controls:

  • Isotype control: Non-specific IgG from same host species at same concentration

  • Secondary-only control: Omit primary antibody but perform all other steps

  • Tissue/cell controls: Use samples with known RPS8 expression profiles

• Cross-method Validation:

  • Verify protein expression using complementary techniques (WB, IHC, IF)

  • Compare protein detection with mRNA levels (qPCR, in situ hybridization)

• Biological Consistency:

  • Evaluate whether staining pattern is consistent with known RPS8 biology

  • For ribosomal proteins like RPS8, expect primarily cytoplasmic localization

Implementing multiple validation approaches provides stronger evidence for specificity than any single method alone, enhancing the reliability of your RPS8-related findings.

What are the optimal storage conditions for RPS8 antibodies?

Proper storage is essential for maintaining antibody performance over time. For RPS8 antibodies, follow these guidelines:

• Long-term Storage:

  • Store at -20°C for long-term stability

  • Most RPS8 antibodies are formulated with 50% glycerol, allowing storage at -20°C without freezing solid

  • Antibodies are typically stable for one year after shipment when stored properly

• Buffer Composition:

  • Proteintech 18228-1-AP: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Boster Bio A07839: PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide

  • The high glycerol content prevents damage from freeze-thaw cycles

• Working Stock Handling:

  • For frequent use, small aliquots can be stored at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles, which cause antibody degradation

  • For Proteintech 18228-1-AP, "aliquoting is unnecessary for -20°C storage"

• Handling Precautions:

  • Bring to room temperature before opening

  • Mix gently by pipetting or finger-tapping; avoid vigorous vortexing

  • Use sterile technique to prevent contamination

  • Note safety considerations: contains sodium azide, which is toxic

• Working Dilution Stability:

  • Prepare diluted working solutions immediately before use

  • Do not store diluted antibody for extended periods

  • If storage of diluted antibody is necessary, add protein carrier (e.g., 0.1% BSA)

Following these storage recommendations will help maintain antibody performance and extend useful shelf life, ensuring consistent results across experiments.

How is RPS8 implicated as a biomarker in cancer research?

Recent studies have identified RPS8 as a promising biomarker in several cancer types:

• Alcohol-associated Hepatocellular Carcinoma (HCC):

  • RPS8 is specifically upregulated in alcohol-associated HCC but not in non-alcohol-associated HCC

  • This specificity makes it a potential diagnostic biomarker for distinguishing alcohol-related liver cancer

  • Gene Set Enrichment Analysis (GSEA) showed that samples with high RPS8 expression had enrichment in RNA polymerase and ribosome pathways

• Colorectal Cancer:

  • Increased RPS8 expression has been documented in colorectal tumors and colon polyps compared to matched normal colonic mucosa

  • This suggests potential utility as a biomarker for colorectal neoplasia progression

• Methodological Approaches for RPS8 Biomarker Research:

  • IHC with anti-RPS8 antibody (1:40 dilution) is the validated method for tissue analysis

  • Quantification using a 0-3 scoring system based on percentage of positive cells

  • Comparison between tumor and adjacent normal tissues establishes differential expression

  • Image analysis software provides objective quantification

• Biomarker Validation Framework:

  • Initial discovery through differential expression analysis

  • Validation in independent cohorts

  • Correlation with clinical outcomes and pathological features

  • Functional studies to understand mechanistic roles

These findings suggest that RPS8 could serve not only as a diagnostic biomarker but potentially as a therapeutic target, particularly in alcohol-associated HCC. The unique expression pattern in specific cancer subtypes indicates its potential utility in precision oncology applications.

What techniques can be used to study RPS8 protein interactions?

Understanding RPS8's interactome is crucial for elucidating its functions beyond ribosome assembly. Several complementary techniques have been successfully employed:

• Yeast Two-Hybrid (Y2H) Assay:

  • Methodology: RPS8 coding sequence is fused to a DNA-binding domain vector (e.g., pGBKT7), while potential interacting partners are fused to an activation domain vector (e.g., pGADT7)

  • Construction: Vectors are created using homologous recombination methods and verified by DNA sequencing

  • This approach has successfully identified interactions between plant RPS8 and viral proteins

• Bimolecular Fluorescence Complementation (BiFC):

  • Methodology: RPS8 and potential interacting partners are fused to complementary fragments of a fluorescent protein

  • Construction: Uses Gateway technology with vectors like pEarleyGate202-YN and pEarleyGate201-YC

  • Advantage: Provides spatial information about where interactions occur within living cells

• Subcellular Localization Studies:

  • Methodology: RPS8 coding sequence is merged into fluorescent protein vectors (e.g., pBin-GFP)

  • Application: Visualizes RPS8 localization and co-localization with potential interacting partners

  • Important for confirming that putative interacting proteins share cellular compartments

• Functional Validation:

  • Gene silencing approaches (e.g., VIGS) to study the functional significance of RPS8 interactions

  • Phenotypic analysis following disruption of specific interactions

  • This integrative approach links interactions to biological functions

When designing RPS8 interaction studies, consider that as a ribosomal protein, RPS8 may have numerous interactions within the ribosome complex. Tools like GSEA can help identify pathways associated with RPS8 interactions, as demonstrated in HCC research where RNA polymerase and ribosome pathways were found to be enriched in samples with high RPS8 expression .

How can Gene Set Enrichment Analysis (GSEA) be applied to RPS8 research?

GSEA provides valuable insights into the pathways and biological processes associated with RPS8 expression patterns. Here's how to effectively implement this approach in RPS8 research:

• Methodological Framework:

  • Sample division: Separate samples into high and low RPS8 expression groups based on median expression level

  • Software: GSEA software (version 4.0.0; Broad Institute) is the standard tool

  • Statistical parameters: Use normalized enrichment score (NES) to quantify association strength

  • Significance thresholds: P<0.01 and NES >1.5 are recommended cutoff values

• Application in Cancer Research:

  • In alcohol-associated HCC studies, GSEA revealed 10 enriched pathways in samples with high RPS8 expression

  • Key pathways included RNA polymerase and ribosome pathways

  • This suggests that RPS8 may influence both transcription and translation in cancer contexts

• Implementation Steps:

  • Generate gene expression data (e.g., RNA-seq, microarray)

  • Rank genes based on correlation with RPS8 expression

  • Use pre-defined gene sets (e.g., KEGG pathways, GO terms)

  • Run GSEA algorithm to identify enriched pathways

  • Visualize results using enrichment plots and heatmaps

• Interpretation Framework:

  • Distinguish between positively and negatively enriched pathways

  • Consider both canonical (ribosome-related) and non-canonical pathways

  • Integrate findings with protein interaction data for a comprehensive understanding

  • Validate key pathways through targeted functional studies

• Extended Applications:

  • Compare pathway enrichment across different cancer types or disease states

  • Identify potential therapeutic targets within enriched pathways

  • Predict functional consequences of RPS8 alterations

This approach has already yielded valuable insights into RPS8's role in alcohol-associated HCC and could be similarly applied to investigate its functions in other biological contexts, potentially revealing novel roles beyond its canonical function in ribosome assembly.

What is known about RPS8's role in translational regulation?

RPS8 functions extend beyond structural roles in the ribosome, with emerging evidence highlighting its importance in translational regulation:

• Rate-limiting Factor in Translation:

  • Research has identified RPS8 as a rate-limiting factor in translational regulation

  • This suggests it may selectively influence the translation of specific mRNAs rather than affecting global protein synthesis uniformly

• Stress Response Functions:

  • RPS8 contributes to cold-adaptability in plants like rice

  • This indicates involvement in stress-responsive translational regulation

  • Such functions may be conserved across different biological systems, including potential roles in mammalian stress responses

• Cancer-Related Translational Control:

  • Upregulation in specific cancer types suggests potential roles in dysregulated translation

  • In alcohol-associated HCC, RPS8 expression correlates with enrichment of RNA polymerase and ribosome pathways

  • This may reflect altered translational programs supporting carcinogenesis

• Methodological Approaches to Study Translational Roles:

  • Ribosome profiling to identify mRNAs whose translation is specifically affected by RPS8 levels

  • Polysome profiling to assess effects on global translation efficiency

  • Reporter assays to study impacts on specific mRNA translation

  • RPS8 silencing followed by proteomic analysis to identify affected proteins

• Research Directions:

  • Investigating whether RPS8 preferentially affects translation of specific mRNA subsets

  • Exploring potential extraribosomal functions in translational control

  • Understanding how post-translational modifications of RPS8 might regulate its function

  • Determining whether RPS8-targeted therapies could modulate disease-specific translational programs

Understanding RPS8's roles in translational regulation may reveal new therapeutic opportunities, particularly in diseases where RPS8 expression is altered, such as alcohol-associated HCC and colorectal cancer.

How should I interpret variations in RPS8 molecular weight on Western blots?

Researchers frequently observe discrepancies between calculated and observed molecular weights for RPS8. Understanding these variations is critical for correct data interpretation:

• Expected vs. Observed Molecular Weights:

  • Calculated molecular weight: 24 kDa (208 amino acids)

  • Observed molecular weights:

    • 25-28 kDa (Proteintech 18228-1-AP)

    • 39 kDa (Boster Bio A07839)

• Potential Explanations for Discrepancies:

  a) Post-translational Modifications:

  • Phosphorylation, ubiquitination, or other modifications can increase apparent molecular weight

  • As a regulatory protein, RPS8 may undergo context-dependent modifications

  b) Sample Preparation Effects:

  • Buffer composition, reducing agent concentration, and heating conditions affect protein migration

  • Incomplete denaturation can cause proteins to run at higher apparent molecular weights

  c) Strong Protein-Protein Interactions:

  • Incomplete dissociation from interaction partners can cause molecular weight shifts

  • As a ribosomal protein, RPS8 forms strong interactions with rRNA and other ribosomal proteins

  d) Antibody Epitope Specificity:

  • Different antibodies recognize different epitopes, potentially detecting different isoforms

  • This explains why different antibodies report different observed molecular weights

• Methodological Approaches to Address Discrepancies:

  a) Validation Strategy:

  • Use multiple antibodies targeting different epitopes of RPS8

  • Include positive controls (e.g., recombinant RPS8 protein)

  • Perform RPS8 knockdown to confirm band identity

  b) Optimization Approaches:

  • Test different sample preparation methods (varying SDS concentration, temperature)

  • Use gradient gels for better resolution

  • Include molecular weight markers that cover the range of interest

When reporting RPS8 Western blot results, always specify the antibody used and the observed molecular weight, as these can vary significantly between different experimental systems and detection methods.

What are common pitfalls in RPS8 immunostaining and how can they be addressed?

Several technical challenges can affect RPS8 immunostaining. Here are methodological solutions for common issues:

• High Background Staining:

  • Problem: Non-specific binding obscuring specific RPS8 signal

  • Solutions:

    • Optimize blocking: Use 5% BSA as validated in RPS8 IHC protocols

    • Increase antibody dilution: Test higher dilutions than the recommended range

    • Extend washing steps: Add additional washes with 0.1% Tween-20 in PBS

    • Use more specific secondary antibodies: Pre-absorbed against serum proteins

• Weak or Absent Signal:

  • Problem: Insufficient detection of RPS8

  • Solutions:

    • Optimize antigen retrieval: Sodium citrate at 100°C has been validated

    • Reduce antibody dilution: Test more concentrated antibody preparations

    • Extend primary antibody incubation: Overnight at 4°C is recommended

    • Use signal amplification systems: Consider tyramide signal amplification

• Variable Staining Across Samples:

  • Problem: Inconsistent RPS8 staining between experiments

  • Solutions:

    • Standardize fixation: Use consistent fixation times and conditions

    • Process all samples simultaneously: Minimize batch effects

    • Include reference standards: Use samples with known RPS8 expression levels

    • Quantify using standardized scoring: Apply the 0-3 scale validated in HCC research

• Non-specific Nuclear Staining:

  • Problem: RPS8 should be primarily cytoplasmic, but nuclear staining may occur

  • Solutions:

    • Verify with subcellular fractionation and Western blot

    • Compare with known patterns: Validated RPS8 antibodies show cytoplasmic staining

    • Perform peptide competition: Nuclear staining that remains after competition may be non-specific

• Methodological Validation Approaches:

  • Include technical controls in every experiment (negative, isotype, secondary-only)

  • Perform dual staining with established ribosomal markers

  • Compare manual and automated scoring for objective quantification

  • Document detailed protocols to ensure reproducibility

By systematically addressing these common pitfalls, researchers can enhance the specificity and reliability of RPS8 immunostaining experiments, yielding more robust and reproducible results.

How can RPS8 expression be accurately quantified in research samples?

Accurate quantification of RPS8 expression requires careful methodological consideration and appropriate analytical techniques:

• Western Blot Quantification:

  • Optimization: Determine linear detection range for RPS8 in your system

  • Normalization: Use total protein normalization (Ponceau S, REVERT) rather than single housekeeping genes

  • Technical considerations:

    • Load equal protein amounts (confirmed by BCA/Bradford assay)

    • Include concentration standards for absolute quantification

    • Use digital image acquisition and analysis software

• Immunohistochemistry Quantification:

  • Validated scoring system: 0 (0-1% positive cells), 1 (1-33%), 2 (34-66%), 3 (67-100%)

  • Image analysis workflow:

    • Capture images at standardized magnification (×200 recommended)

    • Use image analysis software (e.g., Image-Pro Plus) for objective quantification

    • Analyze multiple fields per sample (minimum 3-5)

• RT-qPCR for mRNA Quantification:

  • Reference gene selection: Validate stability across your experimental conditions

  • Primer design: Target regions common to all RPS8 transcript variants

  • Analysis: Use 2^(-ΔΔCt) method with appropriate reference genes

• Statistical Analysis Considerations:

  • Sample size determination: Power analysis based on expected effect size

  • Appropriate statistical tests: t-test, ANOVA, or non-parametric alternatives

  • Multiple testing correction: Benjamini-Hochberg or similar methods for large-scale studies

  • Report both statistical significance and effect size

• Multi-method Integration:

  • Cross-validate findings across different quantification methods

  • Address discrepancies between protein and mRNA levels

  • Consider single-cell approaches for heterogeneous tissues

When reporting RPS8 quantification results, clearly document all methodological details including antibody dilutions, image acquisition parameters, and analysis software settings to ensure reproducibility and facilitate inter-laboratory comparisons.

What approaches can distinguish between specific and non-specific RPS8 antibody binding?

Distinguishing specific from non-specific binding is critical for accurate interpretation of RPS8 antibody results. Implement these methodological approaches:

• Peptide Competition Assay:

  • Methodology: Pre-incubate RPS8 antibody with excess immunizing peptide

  • Interpretation: Specific signals should disappear or significantly diminish

  • Implementation: Run parallel samples with and without peptide competition

  • Note: For Boster Bio A07839, blocking peptide corresponding to the immunogen can be purchased

• Genetic Validation:

  • RNAi approach: Transfect cells with RPS8-targeting siRNA/shRNA

  • CRISPR approach: Generate RPS8 knockout or knockdown cell lines

  • Analysis: Compare staining/signal in normal vs. RPS8-depleted samples

  • Interpretation: Specific signals should decrease proportionally to knockdown efficiency

• Multiple Antibody Validation:

  • Methodology: Test multiple RPS8 antibodies targeting different epitopes

  • Analysis: Compare binding patterns across different antibodies

  • Interpretation: Consistent patterns increase confidence in specificity

  • Example: Compare results from Proteintech 18228-1-AP vs. Thermo Fisher PA5-51052

• Dilution Series Analysis:

  • Methodology: Test a wide range of antibody dilutions beyond manufacturer recommendations

  • Analysis: Plot signal-to-noise ratio against antibody concentration

  • Interpretation: Specific binding typically shows a sigmoidal curve with saturation

  • Implementation: Identify the optimal dilution where specific signal is maximized while background is minimized

• Technical Controls:

  • Secondary-only control: Omit primary antibody to assess secondary antibody background

  • Isotype control: Use non-specific IgG from same host species at same concentration

  • No-sample control: Process without biological sample to identify reagent artifacts

By integrating multiple validation approaches, researchers can build stronger evidence for RPS8 antibody specificity, enhancing the reliability and reproducibility of their experimental findings.