RPS17B Antibody

What is RPS17 protein and what cellular functions does it perform?

RPS17 (ribosomal protein S17) is a component of the small (40S) ribosomal subunit, which forms part of the larger ribonucleoprotein complex responsible for protein synthesis in cells. The canonical human RPS17 protein consists of 135 amino acid residues with a molecular weight of approximately 15.6-16 kDa . It is widely expressed in various tissue types and has subcellular localization in both the nucleus and cytoplasm . As a member of the eukaryotic ribosomal protein eS17 family, RPS17 plays a crucial role in ribosome assembly and function. The protein participates in mRNA binding and forms part of the ribosomal mRNA exit channel . Mutations in the RPS17 gene have been associated with Diamond-Blackfan anemia, a rare genetic disorder characterized by bone marrow failure .

What types of RPS17 antibodies are available for research use?

Several types of RPS17 antibodies have been developed for research applications, including:

These antibodies are available in different formats (e.g., purified IgG, buffered aqueous solution) and have been validated for specific applications, making them suitable for diverse experimental protocols in ribosome biology research .

What are the standard applications for RPS17 antibodies in research?

RPS17 antibodies are validated for several standard research applications:

• Western Blotting (WB): Used to detect RPS17 protein in cell lysates, typically showing a band at 16-19 kDa. Different antibodies have specific recommended dilutions, ranging from 1:500-1:2000 for polyclonal antibodies to 1-5 μg/mL for monoclonal antibodies .

• Immunohistochemistry (IHC): Applied to detect RPS17 in tissue sections, particularly in mouse cerebellum tissue, with recommended dilutions of 1:50-1:500 .

• Immunofluorescence/Immunocytochemistry (IF/ICC): Used to visualize subcellular localization of RPS17 in cultured cells, such as U2OS cells, with recommended dilutions of 1:200-1:800 .

• Immunoprecipitation (IP): Employed in ribosome affinity purification methods to isolate ribosomes and identify ribosome-associated proteins (RAPs) .

• ELISA: Used for quantitative detection of RPS17 protein .

How should researchers optimize Western Blot protocols for RPS17 antibodies?

Optimizing Western Blot protocols for RPS17 antibodies requires careful consideration of several factors:

• Sample Preparation:

  • For cellular samples, verified sources include HEK-293, HeLa, Jurkat, PC-3, and NIH/3T3 cells for polyclonal antibodies .

  • For tissue lysates, protocols may need to be adjusted based on tissue type.

  • Use appropriate lysis buffers that preserve protein integrity while effectively extracting ribosomal proteins.

• Antibody Selection and Dilution:

  • For polyclonal antibodies (e.g., 16267-1-AP), recommended dilutions range from 1:500-1:2000 .

  • For monoclonal antibodies (e.g., SAB1401343), optimal concentration is 1-5 μg/mL .

  • PrecisionAb polyclonal antibodies are recommended at 1:1000 dilution .

• Detection Conditions:

  • Expected molecular weight is 16-19 kDa, with Proteintech antibodies detecting at 16 kDa and Bio-Rad antibodies at approximately 19 kDa in HepG2 cell lysate .

  • Use appropriate molecular weight markers and positive controls (e.g., HEK-293 cell lysate).

• Validation Steps:

  • Include negative controls to confirm specificity.

  • Consider using RNase treatment controls when studying ribosome-associated functions.

  • For quantitative analysis, normalize to appropriate loading controls.

Each antibody should be titrated in the specific testing system to obtain optimal results, as sensitivity may be sample-dependent .

What are the recommended protocols for immunohistochemistry with RPS17 antibodies?

For optimal immunohistochemistry results with RPS17 antibodies:

• Tissue Preparation and Antigen Retrieval:

  • Validated tissues include mouse cerebellum tissue .

  • Primary antigen retrieval recommendation: TE buffer pH 9.0.

  • Alternative method: Citrate buffer pH 6.0 .

• Antibody Incubation:

  • Recommended dilution range for IHC: 1:50-1:500 (for 16267-1-AP) .

  • Optimal incubation time and temperature should be determined empirically.

  • Block non-specific binding sites with appropriate blocking solution.

• Detection System:

  • Use a detection system compatible with rabbit IgG antibodies.

  • Consider signal amplification for low-abundance targets.

• Controls and Validation:

  • Include positive control tissues (mouse cerebellum).

  • Use appropriate negative controls (isotype control or primary antibody omission).

  • Consider dual staining with other ribosomal markers to confirm specificity.

Detailed protocols are available from manufacturers for specific antibodies. For example, Proteintech provides downloadable IHC protocols optimized for their RPS17 antibody (16267-1-AP) .

What factors affect immunofluorescence detection of RPS17 in cellular studies?

Several factors influence the success of immunofluorescence experiments with RPS17 antibodies:

• Cell Type Selection:

  • Validated cell lines include U2OS cells for Proteintech's 16267-1-AP antibody .

  • Different cell types may require protocol adjustments due to varying expression levels.

• Fixation and Permeabilization:

  • Fixation method affects epitope accessibility and antibody binding.

  • Paraformaldehyde (4%) is commonly used, but alternative fixatives may be considered.

  • Permeabilization agents (e.g., Triton X-100, methanol) should be optimized for nuclear/cytoplasmic ribosomal proteins.

• Antibody Dilution and Incubation:

  • Recommended dilutions for IF/ICC: 1:200-1:800 for polyclonal antibodies .

  • Incubation time and temperature should be optimized for signal-to-noise ratio.

• Blocking and Washing Steps:

  • Thorough blocking reduces non-specific binding.

  • Multiple washing steps are essential for reducing background.

• Imaging Parameters:

  • Adjust exposure settings to avoid signal saturation.

  • For co-localization studies with other ribosomal proteins, select compatible fluorophores.

  • Confocal microscopy may provide better resolution for intracellular localization.

Manufacturers provide specific IF protocols that can be adapted to individual laboratory conditions. Proteintech offers downloadable IF protocols optimized for their RPS17 antibody .

How can RPS17 antibodies be used in ribosome affinity purification studies?

RPS17 antibodies have proven valuable for ribosome affinity purification studies, particularly for identifying ribosome-associated proteins (RAPs):

• Experimental Design for Ribosome Isolation:

  • FLAG-tagged RPS17 (eS17-FLAG) can be expressed in cells for immunoprecipitation studies.

  • This approach allows isolation of the small (40S) ribosomal subunit along with associated proteins.

  • MS analysis of eS17-FLAG cells has demonstrated enrichment of both small and large subunits, indicating capture of fully assembled 80S ribosomes .

• Methodology for Affinity Purification:

  • Cytoplasmic isolation is followed by FLAG-immunoprecipitation (IP).

  • Samples are analyzed by LC/MS-MS and evaluated using SAINT analysis.

  • Ribosome interactors are defined based on SAINT score (≥ 0.56) and fold change enrichment (≥ 4) .

• Distinguishing Subunit-Specific Interactions:

  • eS17-FLAG allows identification of 40S-specific interactors.

  • Comparison with large subunit markers (e.g., eL36-FLAG) helps differentiate subunit-specific interactions.

  • For example, Rio2 kinase is specifically identified in eS17-MS data and is known to block the ribosomal mRNA exit channel .

• RNA-Dependent vs. Independent Interactions:

  • RNase treatment helps identify mRNA-dependent RAPs.

  • Approximately 14% of total RAPs lose ribosome interaction upon mRNA digestion .

  • This approach helps classify direct protein-protein interactions versus RNA-mediated associations.

This methodology has led to the identification of approximately 400 proteins that interact with ribosomes, including components of the canonical translation machinery and proteins involved in metabolism, cell cycle, and RNA processing .

What role does RPS17 play in Diamond-Blackfan anemia research and how are antibodies used in this context?

RPS17 has significant relevance in Diamond-Blackfan anemia (DBA) research, with antibodies serving as critical tools:

• Disease Association and Mechanisms:

  • RPS17 gene mutations have been linked to Diamond-Blackfan anemia (DBA4) .

  • DBA is a rare congenital bone marrow failure syndrome characterized by red cell aplasia.

• Research Applications of RPS17 Antibodies in DBA Studies:

  • Expression Analysis: Antibodies help quantify RPS17 levels in patient-derived cells.

  • Structural Studies: Antibodies aid in determining how mutations affect protein localization and ribosome assembly.

  • Functional Analysis: Co-immunoprecipitation with RPS17 antibodies can identify altered protein interactions in disease states.

  • Translational Defects: Western blotting helps assess downstream effects on translation.

• Model Systems and Approaches:

  • Patient-derived cells

  • CRISPR-engineered cell lines carrying RPS17 mutations

  • Animal models of DBA

• Methodological Considerations:

  • When studying patient samples, controls should include healthy donors matched for age and sex.

  • For mutation studies, antibodies recognizing multiple epitopes help ensure detection despite potential structural changes.

  • Quantitative western blot analysis should include normalization to multiple loading controls.

Research in this area contributes to understanding ribosome biogenesis defects in bone marrow failure syndromes and developing potential therapeutic approaches.

How can RPS17 antibodies be employed in studying ribosome heterogeneity and specialized ribosomes?

Recent advances in ribosome biology have revealed ribosome heterogeneity and specialized ribosomes as important regulatory mechanisms. RPS17 antibodies can facilitate this research:

• Tissue-Specific Ribosome Composition:

  • RPS17 antibodies allow comparison of ribosome composition across tissue types.

  • Immunoprecipitation followed by mass spectrometry can identify tissue-specific ribosome-associated factors.

  • Immunohistochemistry can reveal differential expression patterns of RPS17 in various tissues .

• Post-Translational Modifications (PTMs):

  • Specialized antibodies targeting modified forms of RPS17 can detect PTMs.

  • Combined with phospho-specific or ubiquitin-specific antibodies, researchers can investigate how modifications affect ribosome function.

  • Comparison of modified versus unmodified RPS17 levels provides insights into regulatory mechanisms.

• Methodology for Studying Ribosome Heterogeneity:

  • Sucrose Gradient Fractionation: Combined with western blotting using RPS17 antibodies to analyze different ribosomal populations.

  • Proximity Labeling: Using RPS17 as bait to identify cell-type-specific interactors.

  • Single-Cell Analysis: Immunofluorescence with RPS17 antibodies can reveal cell-to-cell variability in ribosome composition.

• Experimental Design Considerations:

  • Include appropriate controls for each cell or tissue type.

  • When comparing ribosome composition, normalize to core ribosomal proteins.

  • Consider complementary approaches such as RNAseq of ribosome-associated mRNAs to correlate with protein-level findings.

This research direction provides insights into how specialized ribosomes contribute to cellular differentiation, stress responses, and disease states.

What are common challenges when using RPS17 antibodies and how can they be addressed?

Researchers working with RPS17 antibodies may encounter several technical challenges:

• Cross-Reactivity Issues:

  • Problem: Antibodies may detect related ribosomal proteins.

  • Solution: Validate specificity using knockout/knockdown controls or peptide competition assays. Select antibodies with demonstrated specificity, such as those detecting a single band of the expected molecular weight (16 kDa for Proteintech, 19 kDa for Bio-Rad) .

• Variable Signal Intensity:

  • Problem: Inconsistent detection across experiments.

  • Solution: Standardize protein loading amounts, optimize antibody dilutions (try ranges from 1:500-1:2000 for WB) , and ensure consistent transfer conditions. Consider using fresh antibody aliquots for critical experiments.

• High Background in Immunostaining:

  • Problem: Non-specific staining obscuring specific signals.

  • Solution: Increase blocking time/concentration, optimize antibody dilution (1:50-1:500 for IHC, 1:200-1:800 for IF) , include additional washing steps, and ensure appropriate negative controls.

• Storage and Stability Issues:

  • Problem: Diminished antibody performance over time.

  • Solution: Store antibodies according to manufacturer recommendations (typically -20°C) . For Proteintech antibodies, they remain stable for one year after shipment when stored properly, and aliquoting is unnecessary for -20°C storage .

• Cell/Tissue-Specific Detection Challenges:

  • Problem: Variation in detection efficiency across sample types.

  • Solution: Verify antibody reactivity with your specific sample type. For example, 16267-1-AP has confirmed reactivity with human and mouse samples , while Bio-Rad's antibody detects RPS17 in HepG2 cell lysate .

• Antigen Retrieval in IHC:

  • Problem: Poor epitope accessibility in fixed tissues.

  • Solution: For Proteintech's RPS17 antibody, use TE buffer pH 9.0 for antigen retrieval, or alternatively, citrate buffer pH 6.0 .

How should researchers validate the specificity of RPS17 antibodies?

Thorough validation is essential for ensuring reliable results with RPS17 antibodies:

• Western Blot Validation:

  • Confirm single band detection at the expected molecular weight (16-19 kDa) .

  • Test multiple cell lines with known RPS17 expression (e.g., HEK-293, HeLa, Jurkat, PC-3, NIH/3T3) .

  • Include positive controls along with experimental samples.

• Genetic Knockdown/Knockout Controls:

  • Use siRNA/shRNA knockdown of RPS17 to confirm signal reduction.

  • CRISPR/Cas9-mediated knockout provides the most stringent control if cell viability permits.

  • Compare signal intensity between control and knockdown/knockout samples.

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide before application.

  • Specific signals should be blocked while non-specific signals remain.

  • For Proteintech's antibody, the immunogen is RPS17 fusion protein Ag9334 .

• Orthogonal Antibody Comparison:

  • Compare results using antibodies targeting different epitopes of RPS17.

  • Consistent detection patterns across antibodies increase confidence in specificity.

  • Consider antibodies from different manufacturers (Proteintech, Bio-Rad, Sigma-Aldrich) .

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP followed by MS to confirm antibody captures RPS17 and associated ribosomal proteins.

  • Expected results should show enrichment of both small and large ribosomal subunits .

• Cross-Species Reactivity Testing:

  • If cross-species applications are needed, validate with samples from each species.

  • Confirmed reactivity has been demonstrated for human and mouse samples .

How can researchers address contradictory results when studying RPS17 in different experimental systems?

When faced with contradictory results across experimental systems:

• Systematic Comparison of Experimental Variables:

  • Cell/Tissue Type Differences: Expression and modification of RPS17 may vary between systems. Analyze expression levels using standardized protocols.

  • Antibody Selection: Different antibodies may recognize distinct epitopes or isoforms. Use multiple validated antibodies (e.g., both Proteintech's polyclonal and Sigma's monoclonal) .

  • Experimental Conditions: Standardize lysis buffers, detection methods, and quantification approaches.

• Analysis of RPS17 Interaction Networks:

  • Use immunoprecipitation to compare RPS17 interaction partners across systems.

  • Consider mRNA-dependent versus independent interactions by performing parallel experiments with RNase treatment .

  • Analyze results using statistical methods such as SAINT analysis with appropriate cutoffs (SAINT score ≥ 0.56, fold change enrichment ≥ 4) .

• Ribosome Assembly State Analysis:

  • Different experimental systems may capture ribosomes in various assembly states.

  • Use sucrose gradient fractionation to analyze ribosome profiles.

  • Compare 40S, 60S, 80S, and polysome distributions between systems.

• Post-Translational Modification Analysis:

  • Contradictory results may stem from differences in RPS17 modifications.

  • Employ 2D gel electrophoresis followed by western blotting to separate modified forms.

  • Use phospho-specific antibodies to detect specific modifications.

• Data Integration Approaches:

  • Employ computational methods to integrate data across systems.

  • Perform Gene Ontology (GO) analysis using appropriate backgrounds (e.g., whole cell proteome) .

  • Create network models of RPS17 interactions to identify system-specific versus conserved functions.

• Experimental Design for Resolving Contradictions:

  • Design paired experiments with internal controls.

  • Use quantitative methods such as TMT labeling for accurate relative protein quantification .

  • Include biological replicates (minimum three) to ensure statistical significance and achieve high correlation between replicates (r=0.93 to 0.99) .

By systematically addressing these factors, researchers can resolve contradictions and develop a more comprehensive understanding of RPS17 biology across different experimental systems.

How can RPS17 antibodies contribute to understanding translational regulation during stress responses?

RPS17 antibodies offer valuable tools for investigating stress-induced translational regulation:

• Stress Granule Association Studies:

  • Immunofluorescence with RPS17 antibodies can track ribosome recruitment to stress granules.

  • Co-localization analysis with stress granule markers provides insights into translational reprogramming.

  • Time-course experiments reveal dynamics of ribosome sequestration during stress recovery.

• Selective mRNA Translation Analysis:

  • Combine RPS17 immunoprecipitation with RNA sequencing to identify mRNAs selectively translated during stress.

  • Compare ribosome-associated transcriptomes across different stress conditions.

  • Analyze how RPS17 modification states correlate with selective translation.

• Methodological Approaches:

  • Polysome profiling combined with western blotting for RPS17 across fractions.

  • Proximity labeling using RPS17 as bait under normal versus stress conditions.

  • FRAP (Fluorescence Recovery After Photobleaching) with fluorescently-tagged antibodies to assess ribosome dynamics.

• Experimental Design Considerations:

  • Include appropriate stress controls and time points.

  • Normalize findings to global translation rates.

  • Consider cell-type specific responses to stress conditions.

This research direction enhances our understanding of how ribosomes contribute to cellular adaptation during environmental challenges and disease states.