RPL17 Antibody Pair

What is RPL17 and what cellular functions does it serve in research contexts?

RPL17 is a component of the 60S ribosomal subunit with a calculated molecular weight of 21 kDa (though it typically appears at 20-23 kDa on western blots) . Beyond its canonical role in protein synthesis, research has revealed RPL17's involvement in multiple cellular processes:

• Ribosomal Function: RPL17 serves as a structural component of the large ribosomal subunit, participating in the ribonucleoprotein complex responsible for protein synthesis .

• RNA Binding: RPL17 demonstrates RNA-binding capabilities, particularly important in viral replication processes. The middle (M) and C-terminal regions are critical for this RNA-binding activity .

• Pathophysiological Roles: RPL17 has been implicated in Diamond-Blackfan anemia (DBA), where mutations lead to defective ribosome formation and erythroid proliferation defects .

• Vascular Smooth Muscle Regulation: Research indicates RPL17 functions as an inhibitor of vascular smooth muscle cell proliferation, with expression differences correlating with intima formation responses across mouse strains .

How should I choose between polyclonal and monoclonal RPL17 antibodies based on my experimental goals?

The selection depends on your specific research objectives:

Polyclonal Antibodies (e.g., 14121-1-AP)

• Advantages: Recognize multiple epitopes, potentially providing stronger signals in applications like western blot and immunohistochemistry .

• Recommended Applications: Effective for western blot (1:2000-1:10000 dilution), immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg protein lysate), and immunohistochemistry (1:50-1:500) .

• Best For: Initial protein detection, immunoprecipitation experiments, and situations where signal amplification is needed.

Monoclonal Antibodies (e.g., 67223-1-Ig)

• Advantages: Provide consistent lot-to-lot reproducibility and higher specificity for a single epitope .

• Recommended Applications: Excellent for western blot (1:5000-1:50000 dilution), immunohistochemistry (1:250-1:1000), and immunofluorescence (1:200-1:800) .

• Best For: Quantitative analyses, experiments requiring high specificity, and applications sensitive to background signal.

What sample preparation techniques optimize RPL17 detection in cellular extracts?

For optimal RPL17 detection in cellular extracts:

• Lysis Buffer Selection: Use RIPA buffer supplemented with protease inhibitors for most applications. For studying RPL17-RNA interactions, consider milder lysis conditions that preserve protein-RNA complexes .

• Sample Processing:

  • For total protein isolation: Lyse cells directly in buffer, sonicate briefly, and centrifuge at 12,000g for 15 minutes at 4°C .

  • For ribosomal fraction isolation: Consider sucrose gradient centrifugation to separate ribosomal subunits.

• Sample Storage: Store protein lysates at -80°C with glycerol (as in commercial antibody formulations with 50% glycerol) .

• Protein Quantification: Use Bradford or BCA assays to normalize loading (typical working range: 20-30 μg total protein per lane for western blot) .

• Positive Controls: HeLa, HepG2, HEK-293, and Jurkat cells consistently show detectable RPL17 expression and serve as excellent positive controls .

What control systems should be incorporated when using RPL17 antibodies for functional studies?

Rigorous experimental designs require appropriate controls:

• Positive Controls:

  • Cell Types: HeLa, HepG2, HEK-293, Jurkat cells, and pancreatic tissues consistently show detectable RPL17 expression .

  • Tissue Types: Pancreatic tissue has demonstrated reliable RPL17 expression for IHC validations .

• Negative Controls:

  • Antibody Specificity: Include secondary antibody-only controls.

  • Knockdown Validation: In RNA interference studies, compare RPL17 knockdown efficiency using both protein detection (western blot) and mRNA quantification (RT-qPCR) as demonstrated in HCV research where knockdown reduced RPL17 mRNA to below 20% .

• Rescue Experiments:

  • Implement siRNA-resistant RPL17 expression vectors to verify phenotypes, similar to approaches used in HCV research where RPL17 knockdown suppressed viral production by >80%, which was significantly restored by introducing siRNA-resistant expression vectors .

• Isotype Controls:

  • For monoclonal antibodies, use matching IgG1 isotype controls (for mouse monoclonals) .

  • For polyclonal antibodies, rabbit IgG fractions serve as appropriate controls .

How can I optimize protocols for studying RPL17-RNA interactions in virus-host systems?

When investigating RPL17-RNA interactions, particularly in viral systems like HCV:

• RNA Immunoprecipitation (RIP):

  • Perform crosslinking with formaldehyde (1%) to stabilize protein-RNA complexes.

  • Immunoprecipitate using 0.5-4.0 μg of anti-RPL17 antibody per 1.0-3.0 mg of protein lysate .

  • Reverse crosslinks and isolate RNA for RT-qPCR analysis.

  • Research demonstrates RPL17 binds specifically to the 3'X region of HCV RNA, with middle and C-terminal regions of RPL17 being essential for this interaction .

• AlphaScreen Assays for quantitative binding analysis:

  • This technique successfully demonstrated reduced binding of RPL17 to HCV RNA with 3'X deletion and specific SLII mutations .

  • Use in vitro synthesized RNA fragments and purified RPL17 protein.

• Mutational Analysis:

  • Implement deletion mutants of RPL17 to identify RNA-binding domains—research showed the 63-residue region on the N-terminal side of RPL17 is not involved in RNA binding, while deletion of middle or C-terminal regions clearly reduced RNA-binding ability .

  • Create RNA mutants (e.g., loop structure modifications as in HCV 3'X/SL2mt3) to identify critical RNA structural elements .

• Co-localization Studies:

  • Use immunofluorescence with RPL17 antibodies (1:200-1:800 dilution) alongside RNA probes.

  • Analyze with confocal microscopy to determine spatial relationships.

What methodologies are effective for investigating RPL17 mutations in hematological disorders?

For investigating RPL17 mutations in conditions like Diamond-Blackfan anemia:

• Genetic Analysis:

  • Perform targeted sequencing of RPL17 exons and splice junctions, as used to identify pathogenic variants (c.452delC; p.(T151Rfs*25)) in DBA patients .

  • Validate variants through bidirectional Sanger sequencing .

• RNA Analysis:

  • Conduct RT-PCR studies to identify alternative splicing products—research identified a variant lacking exon 4 (p.A73_K105del) in DBA patients .

  • Perform quantitative RT-PCR to measure expression levels of wild-type versus mutant transcripts.

• Protein Expression Analysis:

  • Use western blotting with antibodies targeting different regions of RPL17 to detect wild-type and truncated forms .

  • Lymphoblastoid cell lines (LCLs) from patients can be valuable for these analyses .

• Functional Assays:

  • Analyze erythroid cell differentiation using flow cytometry for markers like CD34, CD36, and Glycophorin A (GPA) .

  • Zebrafish models have successfully demonstrated that RPL17/uL22 depletion results in anemia and micrognathia in larvae, providing an in vivo system for mutation studies .

How should I address inconsistent or unexpected RPL17 antibody signals in western blot applications?

When troubleshooting western blot issues with RPL17 antibodies:

• Multiple Bands or Unexpected Molecular Weight:

  • RPL17's calculated molecular weight is 21 kDa, but it typically appears at 20-23 kDa on western blots .

  • If detecting both bands, consider:

    • Post-translational modifications

    • Alternative splicing (as observed in DBA patients with exon 4 skipping)

    • Degradation products (implement fresh protease inhibitors)

• Weak or Absent Signal:

  • Increase antibody concentration—polyclonal antibodies may require 1:2000 dilution rather than 1:10000 .

  • Extend exposure time for chemiluminescence detection.

  • Verify sample integrity through detection of housekeeping proteins.

  • Consider enriching for ribosomal fractions if studying ribosome-incorporated RPL17.

• High Background:

  • Increase blocking time/concentration (5% BSA or milk).

  • Use monoclonal antibodies (e.g., 67223-1-Ig) at higher dilutions (1:5000-1:50000) .

  • Implement additional washing steps with increased TBST volume.

• Sample-Specific Issues:

  • HeLa, HepG2, HEK-293, and Jurkat cells consistently show detectable RPL17 and serve as reliable positive controls .

  • Use 12% SDS-PAGE gels for optimal resolution in the 20-25 kDa range .

What factors contribute to discrepancies when comparing RPL17 detection between different experimental techniques?

Several factors may explain discrepancies between different detection methods:

• Epitope Accessibility Differences:

  • In fixed tissues (IHC/IF), epitope masking may occur, requiring antigen retrieval—suggested methods include TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

  • In native conditions (co-IP), protein conformations may hide epitopes recognized in denatured conditions (western blot).

• Antibody Clone Specificity:

  • C-terminal targeting antibodies (e.g., ABIN653848 targeting AA 156-184) may detect different populations than N-terminal antibodies.

  • When RPL17 mutations affect specific domains, antibodies targeting those regions may show reduced binding, as observed in DBA patients with RPL17 variants .

• Subcellular Localization Effects:

  • Ribosome-incorporated RPL17 versus free protein pools may show different accessibility.

  • RPL17's RNA-binding activity may mask antibody epitopes in RNA-rich cellular compartments .

• Methodology-Specific Variables:

  Technique	Possible Discrepancy Factors	Solution
  Western Blot	Protein denaturation affects epitopes	Try reducing conditions or native PAGE
  IHC/IF	Fixation methods alter protein structure	Test multiple fixation protocols (PFA vs. methanol)
  IP	Antibody may disrupt protein complexes	Use crosslinking approaches
  ELISA	Conformation-specific detection	Validate with capture/detection antibody pairs

How can I distinguish between non-specific binding and authentic detection of RPL17 protein variants?

To confidently distinguish between non-specific signals and true RPL17 variants:

• Genetic Validation:

  • Compare samples with known RPL17 mutations (as in DBA cases with c.261+2T>C variant causing exon 4 skipping) .

  • Create cell lines with tagged RPL17 variants to serve as positive controls.

• Knockdown/Knockout Controls:

  • Implement siRNA knockdown of RPL17 (verification target: <20% remaining expression) .

  • Bands that persist after efficient knockdown likely represent non-specific binding.

• Multiple Antibody Validation:

  • Use antibodies targeting different RPL17 epitopes:

    • C-terminal antibodies (AA 156-184)

    • N-terminal antibodies

    • Full-length antibodies (AA 1-184)

  • True RPL17 variants should be detected by antibodies recognizing retained regions.

• Mass Spectrometry Verification:

  • Excise suspicious bands and perform peptide mass fingerprinting.

  • Compare detected peptides with known RPL17 sequence variants.

What methodological approaches enable investigation of RPL17's role in viral replication complexes?

To investigate RPL17's role in viral replication, particularly with HCV:

• RNA-Protein Interaction Studies:

  • Implement RNA immunoprecipitation (RIP) assays to assess RPL17 binding to viral RNA.

  • Research demonstrated RPL17 binds specifically to the 3'X region within the 3'UTR of HCV genome .

  • RNA-IP assays showed knockdown of RPL17 reduced the amount of HCV RNA co-precipitating with Core to less than 50% .

• Viral Packaging Assessment:

  • Use trans-packaging assays to evaluate viral RNA incorporation into particles.

  • Measure nuclease-resistant viral RNA in culture supernatants—RPL17 knockdown significantly reduced HCV levels in nuclease-resistant fractions .

• Functional Domain Mapping:

  • Create deletion mutants of RPL17 to identify domains essential for viral functions.

  • Research identified that while the 63-residue N-terminal region of RPL17 is not involved in RNA binding, the middle and C-terminal regions are critical .

• Microscopy Approaches:

  • Employ confocal microscopy to assess co-localization between RPL17, viral RNA, and viral proteins.

  • Knockdown studies showed increased spatial distance between HCV Core and dsRNA after RPL17 depletion .

• Rescue Experiments:

  • After RPL17 knockdown, reintroduce siRNA-resistant RPL17 expression vectors.

  • Research showed HCV production was suppressed by >80% with RPL17 knockdown but significantly restored with siRNA-resistant RPL17 expression .

How should researchers design experiments to study the interaction between RPL17 and disease-associated mutations?

For investigating RPL17 mutations in disease contexts like Diamond-Blackfan anemia:

• Patient-Derived Cell Models:

  • Establish lymphoblastoid cell lines (LCLs) from patients with defined RPL17 mutations .

  • Conduct comprehensive phenotyping including:

    • Ribosomal RNA maturation analysis

    • Polysome profiling

    • Translational efficiency measurements

• CRISPR-Based Approaches:

  • Create isogenic cell lines with specific RPL17 mutations.

  • Implement homology-directed repair to introduce patient-specific variants.

  • Compare cellular phenotypes between wild-type and mutant lines.

• Animal Models:

  • Develop zebrafish models—research demonstrated RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae .

  • Use in vivo complementation studies to assess pathogenicity of RPL17 variants .

• Ribosomal Assembly Analysis:

  • Employ sucrose gradient fractionation to analyze ribosome assembly.

  • Research showed LCLs from DBA patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17 .

  • Notably, 10-20% of 60S ribosomal subunits in patient cells contained a short form of 5.8S rRNA (5.8S C), a species marginal in normal cells .

• Translational Profiling:

  • Implement ribosome profiling to assess translational changes.

  • Research showed cells with RPL17 mutations had translational profile changes similar to cells with RPS19 mutations .

What analytical frameworks best capture RPL17's dual functionality in normal ribosomal processes and pathological conditions?

To comprehensively study RPL17's roles in both normal and pathological contexts:

• Integrated Multi-omics Approach:

  • Combine transcriptomics, proteomics, and ribosome profiling data.

  • This approach revealed that while RPL17 variants were not incorporated into ribosomes in DBA patients, the atypical 60S subunits were still actively engaged in translation .

• Comparative Analysis Frameworks:

  • Compare RPL17 function across multiple cellular contexts:

    • Normal ribosomal assembly

    • Virus-infected cells (showed RPL17 facilitates Core-RNA interaction in HCV)

    • Disease states (DBA patients with RPL17 mutations)

    • Vascular smooth muscle cells (where RPL17 expression was >6-fold higher in C3H/F compared to SJL cells)

• Structure-Function Analysis:

  • Correlate structural domains with specific functions:

    • The middle and C-terminal regions of RPL17 are critical for RNA binding

    • The 33 amino acids encoded by exon 4 form a long α-helix within RPL17's structured core, whose absence destabilizes the protein (relevant in DBA)

• Temporal Analysis:

  • Study RPL17 dynamics during:

    • Cellular differentiation (particularly erythroid differentiation)

    • Viral infection cycles

    • Ribosomal stress responses

• Network Analysis:

  • Map RPL17 interactions with:

    • Other ribosomal proteins

    • RNA species (both cellular and viral)

    • Regulatory factors

  • Research identified interaction with Y-box binding protein 1 (YBX1) in HCV replication contexts

What experimental strategies can effectively distinguish between RPL17's canonical ribosomal functions and its non-canonical roles?

To differentiate between canonical and non-canonical functions of RPL17:

• Domain-Specific Mutations:

  • Create RPL17 variants that selectively disrupt:

    • Ribosome incorporation (structural domains)

    • RNA binding (middle and C-terminal regions)

    • Protein-protein interactions

  • Assess the impact on different cellular processes.

• Subcellular Localization Studies:

  • Implement immunofluorescence with RPL17 antibodies (1:200-1:800 dilution) .

  • Co-stain with markers for:

    • Nucleolus (site of ribosome assembly)

    • Cytoplasmic ribosome populations

    • Stress granules and P-bodies

    • Viral replication complexes (in infected cells)

• Temporal Regulation Analysis:

  • Study RPL17 during cellular stress responses when many ribosomal proteins adopt non-canonical functions.

  • Compare RPL17 dynamics during normal and infected cell states.

• Interactome Profiling:

  • Perform quantitative proteomics on RPL17 immunoprecipitates from:

    • Normal cells (capturing canonical interactions)

    • Stressed cells

    • Virus-infected cells (where RPL17 interacts with HCV 3'X RNA and Core protein)

  • Identify condition-specific interaction partners.

• Selective Complementation:

  • In RPL17-depleted systems, reintroduce mutants that retain only canonical or non-canonical functions.

  • Assess which cellular processes are rescued by each construct.

By implementing these advanced approaches, researchers can develop a comprehensive understanding of RPL17's multifaceted roles in normal cellular function and disease pathogenesis.