RPS14 Antibody

What is RPS14 and why is it significant in molecular research?

RPS14 (ribosomal protein S14) is a critical component of the 40S ribosomal subunit in mammalian cells. The protein plays an essential role in ribosome biogenesis and protein translation, making it fundamental to cellular function. RPS14 maintains normal physiological activities by regulating ribosome biosynthesis and translation of important proteins . The gene is particularly significant in research because it maps to the commonly deleted region (CDR) of the 5q- syndrome, connecting it to specific hematological disorders . Its highly conserved nature across species makes it valuable for comparative studies in evolutionary biology and fundamental cellular processes.

What types of RPS14 antibodies are available for research applications?

RPS14 antibodies are available in multiple formats with varying specifications to suit different research needs:

Most commercial RPS14 antibodies are raised against specific amino acid sequences, such as AA 1-151 of human RPS14, and are purified using antigen affinity or Protein A purification methods . The choice between monoclonal and polyclonal antibodies should be guided by experimental requirements for specificity, consistency, and target recognition.

How is species reactivity determined for RPS14 antibodies?

Species reactivity of RPS14 antibodies is established through systematic validation experiments using tissues and cell lines from different organisms. For instance, antibody 16683-1-AP has demonstrated reactivity in human, mouse, and rat samples through positive Western blot detection in HeLa cells, HepG2 cells, mouse liver tissue, and rat liver tissue . Reactivity predictions can also be made based on sequence homology analysis. Immunofluorescence combined with homology analysis has been used to confirm species specificity, as demonstrated in studies with broiler and duck tissues . When selecting an RPS14 antibody, researchers should review the validation data specifically showing reactivity in their species of interest.

What are the optimal dilution ratios for different applications of RPS14 antibodies?

Optimal dilution ratios vary by application and specific antibody. Based on validated protocols:

For 16683-1-AP (Polyclonal):

For 67566-1-Ig (Monoclonal):

These dilutions serve as starting points . It is strongly recommended to titrate the antibody in each testing system to determine optimal conditions for specific experimental setups. Sample-dependent variables like protein expression levels and background signal can significantly impact optimal dilution.

What antigen retrieval methods are most effective for IHC with RPS14 antibodies?

For immunohistochemistry applications with RPS14 antibodies, two primary antigen retrieval methods have demonstrated effectiveness:

• TE buffer at pH 9.0 (primary recommendation)

• Citrate buffer at pH 6.0 (alternative method)

These recommendations are based on successful detection in human liver tissue, human colon tissue (using 16683-1-AP), and human urothelial carcinoma tissue (using 67566-1-Ig) . The selection between these methods may depend on tissue type and fixation conditions. Optimization is particularly important for highly fixed tissues or when detecting low-abundance targets. Researchers should compare both methods if initial results are suboptimal, as RPS14 epitope accessibility can vary between tissue types.

How can I validate the specificity of an RPS14 antibody for my experiments?

Validating RPS14 antibody specificity requires a multi-faceted approach:

• Western blot verification: Confirm detection of a single band at the expected molecular weight (16 kDa for RPS14) .

• Positive and negative controls: Use cell lines with known RPS14 expression levels (e.g., HeLa, HepG2, and A549 cells are positive controls as verified in search results) .

• Knockdown/knockout validation: Demonstrate specificity by comparing detection between RPS14 knockdown/knockout and wild-type samples.

• Cross-reactivity assessment: Test the antibody against related ribosomal proteins when conducting novel investigations.

• Immunoprecipitation followed by mass spectrometry: For definitive validation, identify the precipitated protein is indeed RPS14.

For cross-species applications, homology analysis combined with immunofluorescence testing can verify that observed signals are specific to RPS14 rather than non-specific binding .

What are common troubleshooting strategies for weak or absent signals in Western blots with RPS14 antibodies?

When troubleshooting suboptimal Western blot results with RPS14 antibodies:

• Loading concentration: RPS14 is highly abundant in most cell types, but expression varies. Adjust protein loading to 20-40 μg total protein.

• Antibody concentration optimization: For weak signals, consider using the lower end of the dilution range (e.g., 1:1000 for 16683-1-AP or 1:5000 for 67566-1-Ig) .

• Transfer efficiency: RPS14's small size (16 kDa) can lead to over-transfer. Use PVDF membranes and optimize transfer time (typically shorter than for larger proteins).

• Blocking optimization: Excessive blocking can mask RPS14 epitopes. Test both BSA and non-fat dry milk to determine the optimal blocking agent.

• Sample preparation: Add phosphatase and protease inhibitors to preserve RPS14 integrity. Consider alternative lysis buffers if initial protocols yield poor results.

• Exposure time: Adjust imaging exposure times, as RPS14 signals may require different optimization than larger proteins.

If troubleshooting the polyclonal antibody proves difficult, switching to the monoclonal antibody (67566-1-Ig) may provide more consistent results in certain applications .

How should storage and handling of RPS14 antibodies be optimized for long-term stability?

Proper storage and handling are crucial for maintaining RPS14 antibody performance:

• Storage temperature: Store at -20°C as recommended by manufacturers. Both antibodies (16683-1-AP and 67566-1-Ig) are reported stable for one year after shipment under these conditions .

• Storage buffer composition: The antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which promotes stability .

• Aliquoting considerations: Though the product information states "Aliquoting is unnecessary for -20°C storage," it remains good practice to create single-use aliquots for antibodies used infrequently to prevent freeze-thaw degradation.

• Working solution stability: After dilution, use within 24 hours if stored at 4°C. For longer storage of working solutions, add BSA (0.1-1%) as a stabilizer.

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial contamination that could degrade the product.

• Transport conditions: When transporting between labs or facilities, maintain cold chain using dry ice or sufficient ice packs.

For the 20μl size products, note that they contain 0.1% BSA as an additional stabilizing agent .

How can cross-reactivity between RPS14 and other ribosomal proteins be assessed and mitigated?

Cross-reactivity assessment is particularly important for ribosomal proteins due to structural similarities:

• Sequence alignment analysis: Compare the immunogen sequence (e.g., the fusion protein containing amino acids 1-151 of human RPS14) with other ribosomal proteins to identify potential cross-reactive regions .

• Pre-absorption controls: Pre-incubate the antibody with recombinant RPS14 protein before application to verify signal specificity.

• Multiple antibody verification: Compare results using antibodies raised against different epitopes of RPS14 (e.g., antibodies targeting AA 1-151 versus AA 45-144) .

• Western blot profile analysis: Rigorously examine blots for secondary bands that might indicate cross-reactivity. RPS14 should appear as a single band at 16 kDa .

• Mass spectrometry validation: For definitive cross-reactivity assessment, immunoprecipitate with the RPS14 antibody and analyze by mass spectrometry to identify any co-precipitating proteins.

For advanced applications where absolute specificity is required, consider using monoclonal antibodies like 67566-1-Ig, which typically offer higher specificity than polyclonal alternatives .

How are RPS14 antibodies being utilized in research on hematological disorders?

RPS14 antibodies have become increasingly important in hematological research due to the protein's role in the 5q- syndrome, a subtype of myelodysplastic syndrome (MDS):

• Expression level quantification: Western blot and immunohistochemistry with RPS14 antibodies allow researchers to quantify expression changes in bone marrow samples from MDS patients .

• Cellular localization studies: Immunofluorescence applications reveal altered subcellular distribution of RPS14 in disease states, providing insights into pathological mechanisms .

• Ribosome assembly analysis: RPS14 antibodies help investigate defective ribosome biogenesis in hematological disorders through co-immunoprecipitation of ribosomal assembly factors.

• Therapeutic response monitoring: Tracking RPS14 expression levels following treatment interventions provides a molecular marker for therapeutic efficacy.

• Animal model validation: In studies utilizing mouse models of 5q- syndrome, RPS14 antibodies with cross-reactivity to mouse proteins enable validation of disease mechanisms across species .

These applications collectively contribute to understanding how RPS14 haploinsufficiency leads to the characteristic phenotypes of 5q- syndrome and potentially inform therapeutic strategies targeting ribosomal biogenesis.

What special considerations apply when using RPS14 antibodies in immunofluorescence studies?

Immunofluorescence with RPS14 antibodies requires specific technical considerations:

• Subcellular localization pattern: RPS14 primarily localizes to the nucleolus (site of ribosome biogenesis) and cytoplasm (site of mature ribosomes). This dual localization pattern should be carefully distinguished from non-specific staining .

• Fixation method optimization: Paraformaldehyde (4%) fixation for 15-20 minutes typically preserves RPS14 epitopes while maintaining cellular architecture. Methanol fixation may alter epitope accessibility.

• Background reduction strategies: For polyclonal antibodies like 16683-1-AP or ABIN6147198, extend blocking times (1-2 hours) and use higher concentrations of blocking agents to reduce non-specific binding .

• Co-localization controls: Include co-staining with established nucleolar markers (e.g., fibrillarin) to validate RPS14 nucleolar localization.

• Cell type considerations: RPS14 expression patterns vary between cell types. HepG2 cells have been specifically validated for immunofluorescence/ICC with the 67566-1-Ig antibody .

• Optimal antibody dilution: For IF/ICC applications with 67566-1-Ig, the recommended dilution range is 1:400-1:1600, which may require optimization for specific cell types .

Successful immunofluorescence imaging of RPS14 has been achieved in multiple cell lines including HepG2 cells, providing valuable insights into ribosomal protein dynamics in different cellular compartments .

How can RPS14 antibodies be utilized in studies of non-mammalian model organisms?

Applying RPS14 antibodies to non-mammalian models requires careful consideration of evolutionary conservation and cross-reactivity:

• Cross-species reactivity assessment: While the primary reactivity of most commercial RPS14 antibodies is documented for human, mouse, and rat samples, cited reactivity extends to zebrafish and even Arabidopsis in some cases . This suggests utility in evolutionary studies.

• Sequence homology analysis: Before application in non-mammalian models, align the target species' RPS14 sequence with the immunogen sequence to predict reactivity. Ribosomal proteins are highly conserved, enhancing the likelihood of cross-reactivity.

• Validation in non-mammalian tissues: Studies have demonstrated the application of RPS14 antibodies in avian models, specifically broilers and ducks . These findings suggest potential broader applications in other vertebrate models.

• Application-specific optimization: When applying to non-mammalian species, more extensive antibody titration may be necessary, typically starting at higher concentrations than those used for mammalian samples.

• Controls for non-mammalian studies: Include known positive mammalian samples alongside non-mammalian samples to benchmark reactivity and signal quality when expanding to new species.

The successful preparation and application of RPS14 polyclonal antibodies in broiler research demonstrates the feasibility of extending RPS14 immunological studies beyond traditional mammalian models .

How are RPS14 antibodies contributing to research on broiler ascites syndrome?

Recent research has utilized RPS14 antibodies to investigate broiler ascites syndrome (BAS), revealing previously unrecognized connections between ribosomal proteins and poultry pathophysiology:

• Expression pattern analysis: Western blotting with RPS14 antibodies has demonstrated that BAS significantly reduces RPS14 expression levels in affected birds, establishing a molecular correlation with the disease state .

• Tissue-specific investigations: Immunofluorescence combined with homology analysis revealed that RPS14 expression in key tissues of broilers and ducks is more significant compared to other species, suggesting specialized functions in avian physiology .

• Species-specific antibody development: The successful preparation of rabbit anti-chicken RPS14 serum with significant species specificity has enabled targeted investigation of avian-specific aspects of RPS14 biology .

• Pathophysiological insights: The observation that BAS affects RPS14 expression provides a novel entry point for understanding the molecular basis of this economically significant poultry disease .

• Molecular marker potential: The relationship between RPS14 expression and BAS suggests the protein could serve as a biomarker for disease progression or treatment efficacy.

These findings highlight how RPS14 antibodies can bridge basic ribosomal biology research with applied veterinary science, potentially informing both fields .

What considerations apply when selecting between monoclonal and polyclonal RPS14 antibodies for complex experimental designs?

The choice between monoclonal and polyclonal RPS14 antibodies significantly impacts experimental outcomes:

For complex experimental designs:

• Multi-species studies: Polyclonal antibodies may offer advantages due to recognition of conserved epitopes across species, though specificity should be validated .

• Conformational state detection: Polyclonal antibodies can recognize proteins in different conformational states, which may be advantageous for certain applications .

• Co-immunoprecipitation: Monoclonal antibodies typically provide cleaner IP results with fewer non-specific interactions .

• Quantitative analyses: Monoclonal antibodies offer greater consistency for comparative and longitudinal studies where precise quantification is essential .

• Post-translational modification studies: If the modification impacts the epitope recognized by a monoclonal antibody, signal could be lost entirely, whereas polyclonal antibodies might still detect the protein via other epitopes .

For critical applications, validating key findings with both antibody types provides the strongest experimental confirmation.

How can RPS14 antibodies be integrated into multi-omics research approaches?

Integrating RPS14 antibodies into multi-omics research frameworks enables more comprehensive understanding of ribosomal biology:

• Proteomics integration: RPS14 immunoprecipitation followed by mass spectrometry can identify novel protein interactions within and beyond the ribosomal complex, revealing unexpected functional connections.

• Transcriptomics correlation: Combining RPS14 protein detection (via antibodies) with RNA-seq data allows correlation between RPS14 protein levels and global translational outputs, potentially identifying genes particularly sensitive to RPS14 abundance.

• Chromatin interactions: ChIP-seq applications using RPS14 antibodies can explore potential extraribosomal functions, including potential roles in transcriptional regulation or chromatin organization.

• Spatial transcriptomics integration: Immunofluorescence with RPS14 antibodies can be combined with in situ RNA detection to correlate local RPS14 protein concentration with region-specific translation activity.

• Single-cell multi-omics: RPS14 antibodies compatible with flow cytometry enable isolation of cells with varying RPS14 expression levels for subsequent multi-omics analysis, revealing cell-to-cell variability in ribosome composition and function.

These integrated approaches promise to reveal new dimensions of RPS14 biology beyond its structural role in the 40S ribosomal subunit, potentially identifying novel therapeutic targets for RPS14-associated disorders.

What methodological adaptations are required when using RPS14 antibodies for high-throughput screening applications?

Adapting RPS14 antibodies for high-throughput screening necessitates specific methodological considerations:

• Automation compatibility: For plate-based assays, the 67566-1-Ig monoclonal antibody may offer superior performance due to its wider working dilution range (1:5000-1:50000 for WB) and greater lot-to-lot consistency .

• Signal-to-noise optimization: High-throughput applications require exceptional signal clarity. Pre-absorption of antibodies with non-specific proteins and extended blocking steps may be necessary to minimize background.

• Miniaturization considerations: When adapting to microwell formats, increase antibody concentrations by 1.5-2× compared to standard protocols to compensate for increased surface-to-volume ratios.

• Detection system selection: For high-throughput fluorescence-based detection, select secondary antibodies with bright, photostable fluorophores that resist photobleaching during automated image acquisition.

• Positive control implementation: Include established cell lines with known RPS14 expression levels (e.g., HeLa, HepG2, A549) as internal controls on each plate to normalize results across batches .

• Quantification standardization: Develop a standard curve using recombinant RPS14 protein to enable absolute quantification across plates and experimental runs.

• Cross-platform validation: Confirm hits from antibody-based screens with orthogonal methods such as RNA-seq or ribosome profiling to increase confidence in screening results.

These adaptations enable reliable application of RPS14 antibodies in drug discovery and genetic screening pipelines focused on ribosomal biology and related disorders.