RPS16 Antibody

What is RPS16 and why is it a significant research target?

RPS16 (Ribosomal Protein S16) is a 16 kDa protein component of the small 40S ribosomal subunit. It belongs to the ribosomal protein S9P family and is located in the cytoplasm where it can undergo acetylation . The significance of RPS16 as a research target stems from its critical role in fundamental cellular processes including rRNA processing, translational elongation, initiation, and termination via its RNA binding activity . Additionally, recent studies have identified RPS16 as a substrate of USP1 (ubiquitin-specific peptidase 1), implicating it in hepatocellular carcinoma progression and metastasis . Furthermore, RPS16 was previously identified as an oncoprotein in breast cancer and gliomas by mediating resistance to doxorubicin or activating the PI3K/AKT/Snail pathway .

What are the key specifications to consider when selecting an RPS16 antibody?

When selecting an RPS16 antibody for research, consider these critical specifications:

The antibody selection should align with your specific experimental approach. For instance, if performing Western blot analysis, ensure the antibody is validated for this application with recommended dilutions (typically 1:500-1:2000) . For immunohistochemistry applications, consider antibodies validated at dilutions between 1:20-1:200 .

What is the difference between polyclonal and monoclonal RPS16 antibodies in research applications?

The choice between polyclonal and monoclonal RPS16 antibodies significantly impacts experimental outcomes:

Polyclonal RPS16 Antibodies:

• Recognize multiple epitopes on the RPS16 protein, increasing detection sensitivity

• Available from various hosts including rabbit (e.g., 15603-1-AP, NBP1-80025) and mouse (e.g., H00006217-A01)

• Typically generated against full-length RPS16 or synthetic peptides

• Provide robust signal in applications like Western blot and IHC

• Variation between lots may occur due to the nature of polyclonal production

• Ideal for initial protein detection or when protein abundance is low

Monoclonal RPS16 Antibodies:

For critical research requiring high reproducibility, monoclonal antibodies like EPR11755 may be preferable, while for detection of native or denatured RPS16 in diverse applications, polyclonal antibodies provide versatility and sensitivity .

How does USP1-dependent deubiquitination affect RPS16 protein stability in cancer research models?

USP1 (ubiquitin-specific peptidase 1) plays a critical role in regulating RPS16 protein stability through deubiquitination, which has significant implications for cancer biology. Recent research has elucidated this regulatory mechanism:

In hepatocellular carcinoma (HCC) models, USP1 has been identified as a key regulator of RPS16 protein levels. Both pharmacological inhibition (using ML323) and genetic ablation (via RNAi) of USP1 significantly reduced RPS16 protein expression without affecting other ribosomal proteins like RPS4X and RPS18 . This demonstrates the specificity of the USP1-RPS16 interaction.

The proteasome-dependent degradation of RPS16 was confirmed through experiments with Bortezomib (BTZ), a specific proteasome inhibitor. Treatment with BTZ increased RPS16 protein expression in HepG2 cells, confirming that RPS16 is degraded through the ubiquitin-proteasome pathway when not protected by USP1-mediated deubiquitination .

The USP1-RPS16 interaction involves specific binding domains, which has been investigated through truncated mutants of USP1. Co-immunoprecipitation assays and cellular immunofluorescence studies provided both biochemical and morphological evidence of this protein interaction .

This USP1-RPS16 regulatory axis has functional consequences in cancer, as RPS16 stability affects cellular proliferation and metastasis in HCC models. This suggests targeting the USP1-RPS16 interaction could be a potential therapeutic strategy for HCC treatment .

What are the challenges in validating RPS16 antibody specificity against its multiple pseudogenes?

Validating RPS16 antibody specificity presents significant challenges due to the presence of multiple processed pseudogenes in the genome . This scenario creates several complex issues for researchers:

Cross-reactivity concerns:

• The human genome contains numerous processed pseudogenes of RPS16, which share high sequence similarity with the functional gene

• These pseudogenes can potentially be transcribed and even translated in certain cellular contexts

• Antibodies may inadvertently detect proteins encoded by pseudogenes, leading to false positives

Validation strategies to overcome these challenges:

• Genetic knockdown/knockout validation: Utilizing RNAi (like the siRNAs targeting human RPS16 mentioned in ) to reduce expression of authentic RPS16 and confirm antibody specificity

• Epitope mapping: Detailed knowledge of the epitope recognized by the antibody can help predict potential cross-reactivity

• Multiple detection methods: Combining antibody-based detection with mass spectrometry or other techniques that can distinguish between closely related proteins

• Careful immunogen selection: Antibodies raised against unique regions of RPS16 are less likely to cross-react with pseudogene products

For example, the recombinant monoclonal antibody EPR11755 may offer advantages in specificity due to its singular epitope recognition, whereas polyclonal antibodies might recognize conserved regions present in pseudogene products.

Researchers should be particularly vigilant when studying RPS16 in contexts where pseudogene expression might be altered, such as in cancer cells or during cellular stress responses.

How do post-translational modifications of RPS16, particularly acetylation, affect antibody recognition in different experimental contexts?

Post-translational modifications (PTMs) of RPS16, especially acetylation, can significantly impact antibody recognition and experimental outcomes. This creates an important consideration for researchers:

Impact of acetylation on antibody recognition:

• RPS16 can be acetylated in the cytoplasm , which modifies its structural and biochemical properties

• Acetylation can mask or alter epitopes recognized by certain antibodies

• The extent of RPS16 acetylation may vary between cell types, tissues, and physiological/pathological conditions

Experimental considerations:

• Antibody selection: When studying acetylated forms of RPS16, researchers should select antibodies that either recognize regions unaffected by acetylation or specifically target the acetylated form

• Sample preparation: Preservation of PTMs during sample preparation is critical; certain lysis buffers or fixation protocols may affect the acetylation status

• Control experiments: Include appropriate controls to validate whether the antibody recognition is affected by acetylation status

Research strategies:

• Comparing multiple antibodies: Use antibodies targeting different epitopes of RPS16 to ensure comprehensive detection

• Modification-specific antibodies: Consider using acetylation-specific antibodies when studying this specific PTM

• Biochemical verification: Complement antibody-based detection with mass spectrometry to confirm PTM status

When working with acetylated RPS16, researchers should note that most commercial antibodies (such as those listed in search results - ) do not explicitly state whether they recognize the acetylated form. Therefore, preliminary validation experiments are recommended when studying specific PTMs of RPS16.

What are the optimal protocols for using RPS16 antibodies in Western blot applications?

Optimizing Western blot protocols for RPS16 antibodies requires careful consideration of several technical factors:

Standard Protocol for RPS16 Western Blotting:

• Sample Preparation:

  • Lyse cells in appropriate buffer (RIPA buffer with protease inhibitors works well)

  • For HCC cell lines like HepG2, protocols from study have been validated

  • Load 25μg of protein per lane as demonstrated in validated Western blots

• Gel Electrophoresis:

  • Use 12-15% SDS-PAGE gels (optimal for resolving the 16 kDa RPS16 protein)

  • Include molecular weight markers covering the 10-20 kDa range

• Transfer and Blocking:

  • Transfer to PVDF or nitrocellulose membrane

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

• Primary Antibody Incubation:

  • Dilution ranges vary by antibody source:

    • For rabbit polyclonal (15603-1-AP): 1:500-1:1000

    • For rabbit polyclonal (NBP1-80025): 1.0 μg/ml

    • For mouse polyclonal (H00006217-A01): 1:500-1:2000

  • Incubate overnight at 4°C for optimal results

• Detection:

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse)

  • Expected band: 16 kDa (confirmed observed molecular weight)

  • Note: Some antibodies may detect additional bands due to post-translational modifications

Critical Optimization Parameters:

• Lysate quantity: Titrate between 15-30μg of total protein to determine optimal loading

• Blocking agent: If background is high, consider BSA instead of milk

• Antibody concentration: Lower concentrations may improve specificity but decrease sensitivity

• Exposure time: RPS16 is generally abundant in cells, so shorter exposures may be sufficient

Validation Controls:

• Positive control: Jurkat cell lysate has been validated for Western blot applications

• Negative control: Consider using siRNA knockdown of RPS16 as demonstrated in

What are the critical considerations for immunohistochemistry applications with RPS16 antibodies?

Successful immunohistochemistry (IHC) with RPS16 antibodies requires attention to several critical factors:

Optimized IHC Protocol for RPS16:

• Tissue Preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections (4-6μm thickness)

  • Fresh frozen sections also applicable for certain antibodies

• Antigen Retrieval (Critical Step):

  • Heat-induced epitope retrieval (HIER) is recommended

  • Two validated buffer options:

    • TE buffer pH 9.0 (preferred for many RPS16 antibodies)

    • Citrate buffer pH 6.0 (alternative method)

  • Microwave or pressure cooker heating for 10-20 minutes

• Blocking and Primary Antibody:

  • Block with 5-10% normal serum from secondary antibody host species

  • Primary antibody dilutions vary significantly:

    • Rabbit polyclonal (15603-1-AP): 1:20-1:200

    • Rabbit polyclonal (NBP1-80025): 1:10-1:500

    • HPA064222: 1:200-1:500

  • Incubate overnight at 4°C for optimal staining

• Detection System:

  • Use appropriate HRP-polymer or biotin-based detection system

  • DAB chromogen typically provides good contrast

  • Counterstain with hematoxylin for nuclear visualization

Tissue-Specific Considerations:

• Cancer tissues: Higher expression of RPS16 may be observed, particularly in HCC and breast cancer

• Normal tissues: Cytoplasmic staining pattern is expected

• Controls: Human breast cancer tissue has been validated as a positive control

Validation and Interpretation:

• RPS16 primarily shows cytoplasmic localization

• For quantification, Image J software has been successfully used

• When studying RPS16 in cancer tissues, compare with matched normal tissue controls

• For dual staining, consider combining with Ki67 for proliferation studies, as validated in

An important note from published research: In HCC xenografts, immunohistochemistry assays using anti-RPS16 antibodies have been successfully combined with anti-Ki67 to correlate RPS16 expression with proliferation .

How can researchers effectively optimize immunofluorescence protocols for RPS16 visualization in subcellular localization studies?

Optimizing immunofluorescence (IF) protocols for RPS16 subcellular localization requires attention to preservation of cellular architecture and specific detection parameters:

Detailed IF Protocol for RPS16 Localization:

• Cell Preparation:

  • Validated cell lines: MCF-7 , HeLa, HepG2

  • Grow cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.2-0.5% Triton X-100 (5-10 minutes)

• Blocking and Primary Antibody:

  • Block with 2-5% BSA or normal serum in PBS (1 hour at room temperature)

  • Primary antibody dilutions:

    • Rabbit polyclonal (15603-1-AP): 1:10-1:100

    • Polyclonal antibodies from Abbexa: 1:20-1:200

    • Monoclonal antibody (EPR11755): Demonstrated efficacy in IF

  • Incubate overnight at 4°C in humid chamber

• Secondary Antibody and Counterstaining:

  • Use fluorophore-conjugated secondary antibodies (Alexa Fluor series recommended)

  • Nuclear counterstain: DAPI or Hoechst (blue)

  • Cytoskeletal counterstain (optional): Phalloidin (red/green)

  • Mount with anti-fade mounting medium

• Imaging Parameters:

  • Confocal microscopy offers superior resolution for subcellular localization

  • Z-stack imaging recommended to capture complete cytoplasmic distribution

  • Exposure settings: Start with conservative settings to avoid overexposure

Subcellular Localization Insights:

• RPS16 shows primarily cytoplasmic localization with enrichment in regions of active translation

• Cellular immunofluorescence assays have revealed co-localization between USP1 and RPS16 (shown as yellow/orange areas in merged images)

• Both pharmacological (ML323) and genetic (RNAi) inhibition of USP1 result in decreased RPS16 staining intensity

Multiplexing Strategies:

• Co-staining with nucleolar markers (e.g., fibrillarin) can help distinguish ribosome biogenesis localization

• ER markers (e.g., calnexin) can highlight RPS16 association with rough ER

• USP1 co-localization provides insights into regulatory mechanisms

Controls and Validation:

• Peptide competition controls can verify antibody specificity

• siRNA knockdown of RPS16 serves as a negative control

• Include unstained and secondary-only controls to assess background

Cellular immunofluorescence studies have not only confirmed the interaction between USP1 and RPS16 but also demonstrated that inhibition of USP1 leads to reduced RPS16 levels, supporting the regulatory role of USP1 in controlling RPS16 stability .

What are the common pitfalls in RPS16 antibody-based experiments and how can researchers overcome them?

Researchers working with RPS16 antibodies may encounter several challenges that can impact experimental outcomes:

Common Pitfalls and Solutions:

Antibody Validation Strategies:

• Orthogonal validation: Combine antibody-based detection with orthogonal methods (e.g., mass spectrometry)

• Genetic validation: Use RPS16 siRNA knockdown as demonstrated in the hepatocellular carcinoma study

• Independent antibody validation: Compare results using antibodies targeting different epitopes

• Recombinant expression: Use tagged recombinant RPS16 as a positive control

Special Considerations:

• Ribosomal context: RPS16 function occurs within the ribosomal complex; some epitopes may be masked in intact ribosomes

• USP1 interaction: USP1 inhibition reduces RPS16 levels, which may affect detection sensitivity

• Cell type specificity: Expression levels and post-translational modifications may vary between cell types

How can researchers effectively use RPS16 antibodies in co-immunoprecipitation studies to investigate protein interactions?

Co-immunoprecipitation (Co-IP) with RPS16 antibodies provides valuable insights into its protein interaction network. Based on the search results, particularly the successful Co-IP experiments involving USP1 and RPS16 , here's a comprehensive methodology:

Optimized Co-IP Protocol for RPS16:

• Antibody Selection:

  • Validated antibodies for IP: EPR11755 (ab177951) has been successfully used in IP from fetal liver lysate

  • Alternative: Use tagged RPS16 (HA-RPS16) for pull-down experiments

• Sample Preparation:

  • Harvest cells at 70-80% confluence

  • Lyse cells in non-denaturing lysis buffer (recommended composition based on ):

    • 50 mM Tris-HCl (pH 7.4)

    • 150 mM NaCl

    • 1% NP-40 or 0.5% Triton X-100

    • 1 mM EDTA

    • Protease inhibitor cocktail

  • Clear lysate by centrifugation (13,000 rpm for 10 minutes at 4°C)

• Dynabeads Coupling and IP:

  • Use an Antibody Coupling Kit (e.g., #14311D, Invitrogen)

  • Incubate dynabeads with RPS16 antibody for 16-24 hours

  • Incubate antibody-coupled beads with cell lysate for 1-2 hours at 4°C

  • Wash beads 3-5 times with lysis buffer

• Elution and Analysis:

  • Elute protein complexes with SDS sample buffer

  • Heat at 70°C for 10 minutes

  • Separate complexes by centrifugation (13,000 rpm for 2 minutes)

  • Analyze by Western blotting or mass spectrometry

Demonstrated Protein Interactions:

• USP1 has been confirmed as an interaction partner through Co-IP experiments

• The USP1-RPS16 interaction involves specific binding domains, which were mapped using truncated mutants of USP1

Controls and Validation:

• Input control: 5-10% of pre-cleared lysate

• IgG control: Non-specific IgG from the same species as the primary antibody

• Reciprocal IP: Confirm interactions by IP with antibodies against the suspected binding partner (e.g., USP1)

• Truncation mutants: Can help map interaction domains as demonstrated with USP1 truncated mutants

Advanced Analysis:

• Mass spectrometry of co-immunoprecipitated proteins can reveal novel interaction partners

• To distinguish direct and indirect interactions, consider using purified recombinant proteins

• Molecular dynamics simulation can provide additional evidence for protein-protein interactions, as demonstrated for the USP1-RPS16 complex

This protocol is based on successful Co-IP experiments that identified RPS16 as a substrate of USP1 and elucidated their interaction domains .

What approaches can researchers use to study RPS16 roles in disease models beyond standard antibody applications?

While antibodies are valuable tools for studying RPS16, researchers can employ complementary approaches to gain deeper insights into its roles in disease models:

Genetic Manipulation Approaches:

• RNA interference (RNAi):

  • siRNAs targeting human RPS16 have been successfully used to knock down expression

  • Commercial siRNAs are available (e.g., sc-97200, Santa Cruz)

  • Transfection protocols using lipofectamine RNAiMax have been validated

• CRISPR/Cas9 genome editing:

  • For partial or complete knockout of RPS16

  • For introducing specific mutations or tags at the endogenous locus

  • Consider inducible systems as complete knockout may not be viable

• Yeast genetic models:

  • Yeast strains with RPS16 mutations have been developed (strain Y-318 with pGAL-RPS16A)

  • Allow study of conserved functions in a simpler model organism

  • Enable systematic mutagenesis to identify functional residues

Functional Analysis Techniques:

• Ribosome profiling:

  • Assess impact of RPS16 alterations on global translation

  • Identify specific mRNAs affected by RPS16 dysfunction

• Polysome analysis:

  • Evaluate effects on ribosomal assembly and function

  • Has been validated for RPS16 studies in yeast grown in YEPD medium

• Proteomics approaches:

  • Quantitative proteomics to identify changes in protein expression

  • Protein turnover studies to assess translational efficiency

Disease-Specific Models:

• Cancer models:

  • Xenograft models as used in HCC studies

  • Patient-derived organoids for personalized medicine approaches

  • Correlation of RPS16 expression with clinical outcomes

• Molecular interaction studies:

  • Beyond the USP1-RPS16 axis in HCC

  • Investigation of RPS16 role in the PI3K/AKT/Snail pathway

  • Molecular dynamics simulation to predict interactions

• Therapeutic targeting approaches:

  • Compounds that inhibit the USP1-RPS16 interaction

  • Antisense oligonucleotides targeting RPS16 mRNA

  • Small molecules affecting RPS16 post-translational modifications

Innovative Disease-Relevant Assays:

• Cell migration and invasion assays:

  • Given RPS16's role in promoting metastasis in HCC

  • Wound healing and transwell invasion assays

• Drug resistance studies:

  • RPS16 has been implicated in doxorubicin resistance

  • Combinatorial drug treatments and resistance development

• Patient sample analysis:

  • Correlation between USP1 and RPS16 protein levels using Pearson correlation analysis

  • Kaplan-Meier survival analysis in relation to RPS16 expression

The research on USP1-dependent RPS16 protein stability in HCC demonstrates how combining multiple approaches (biochemical, cellular, animal models, and clinical correlations) can provide comprehensive insights into RPS16's roles in disease pathogenesis .