RPS13 Antibody

What is RPS13 protein and why is it significant in cellular research?

RPS13, also known as 40S ribosomal protein S13, belongs to the ribosomal protein S15P family and functions as a critical component of the 40S ribosomal subunit. Ribosomes, which catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit composed of approximately 80 structurally distinct proteins and 4 RNA species. RPS13 is located primarily in the cytoplasm and has been shown to bind to 5.8S rRNA in rat models, similar to its E. coli ortholog that functions in early ribosome assembly. The gene is notably co-transcribed with two U14 small nucleolar RNA genes located in its third and fifth introns, demonstrating an interesting genomic organization characteristic of ribosomal protein genes. Additionally, like other ribosomal protein genes, RPS13 has multiple processed pseudogenes dispersed throughout the genome, which presents interesting challenges for genetic analysis and targeting specificity .

What are the molecular characteristics of RPS13 that researchers should know?

Researchers working with RPS13 should be aware of its key molecular parameters. The protein has a calculated molecular weight of 17 kDa, though it is sometimes observed at 14 kDa and 17 kDa in experimental conditions such as Western blot analyses. Its GenBank accession number is BC006772, NCBI gene ID is 6207 (human), and UniProt ID is P62277. The human RPS13 protein sequence contains several conserved regions, particularly in the middle region (amino acids 79-151), which includes the sequence "ILRILKSKGL APDLPEDLYH LIKKAVAVRK HLERNRKDKD AKFRLILIES" that has been used as an immunogen for antibody production. This region shows high conservation across species, with 100% identity in cow, guinea pig, horse, human, mouse, rabbit, and rat, 93% in dog, and 86% in yeast, explaining the broad cross-reactivity of many anti-RPS13 antibodies across diverse experimental models .

What applications are RPS13 antibodies validated for in the laboratory?

RPS13 antibodies have been validated for multiple experimental applications, with varying recommended dilutions for optimal results:

Researchers should note that optimal dilutions may be sample-dependent and require titration in specific experimental systems to achieve maximum signal-to-noise ratios. Published research has particularly validated Western blot applications, with at least four published studies demonstrating successful use of anti-RPS13 antibodies in this context .

What are the optimal conditions for RPS13 detection in Western blotting?

For optimal RPS13 detection in Western blotting, researchers should consider several methodological factors. Most commercially available RPS13 antibodies (typically rabbit polyclonal) work effectively at dilutions between 1:1000 and 1:4000 in standard blocking solutions. When preparing samples, researchers should be aware that RPS13 has been observed at both 14 kDa and 17 kDa molecular weights, with the calculated molecular weight being 17 kDa. For cell and tissue lysates, standard RIPA or NP-40 buffers with protease inhibitors are appropriate. MCF-7 cells, mouse uterus tissue, and rat uterus tissue have been confirmed as positive controls for Western blot applications. Following transfer to membrane, overnight incubation with primary antibody at 4°C typically yields optimal results. It's important to note that because RPS13 is highly conserved across species, antibodies frequently show cross-reactivity with human, mouse, rat, and other mammalian samples, making these reagents versatile tools for comparative studies across model organisms .

How should sample preparation be optimized for immunohistochemical detection of RPS13?

Optimizing immunohistochemical detection of RPS13 requires careful consideration of sample preparation and antigen retrieval methods. For formalin-fixed, paraffin-embedded tissues, RPS13 epitopes may be masked and require specific antigen retrieval techniques. The recommended protocol suggests antigen retrieval with TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative. Human colon cancer tissue has been validated as a positive control for RPS13 immunohistochemical detection. The recommended dilution range for IHC applications is 1:50-1:500, with optimal concentrations depending on tissue type and fixation conditions. Researchers should implement appropriate positive and negative controls, including isotype controls and competing peptide blocking, to confirm binding specificity. For chromogenic detection systems such as DAB, hematoxylin counterstaining provides excellent contrast for visualizing RPS13 localization within cellular compartments. Importantly, researchers should validate antibody specificity through Western blot or immunoprecipitation before proceeding with IHC applications to ensure target-specific binding .

What cross-reactivity considerations should be addressed when selecting RPS13 antibodies?

When selecting RPS13 antibodies, researchers must carefully consider cross-reactivity profiles to ensure experimental validity. RPS13 is highly conserved across species, with predicated reactivity showing 100% sequence homology in cow, guinea pig, horse, human, mouse, rabbit, and rat, 93% in dog, 86% in yeast, and 100% in zebrafish for many commercially available antibodies. This high conservation makes RPS13 antibodies useful for cross-species studies but necessitates careful validation when species specificity is required. Additionally, researchers should be aware that as is typical for ribosomal proteins, there are multiple processed pseudogenes of RPS13 dispersed throughout the genome that may share sequence homology with the functional protein. When absolute specificity is required, researchers should select antibodies raised against unique regions of RPS13 or validate specificity through knockout/knockdown controls. Furthermore, some antibodies may cross-react with similar ribosomal proteins in the S15P family, necessitating careful epitope selection and validation through competing peptide blocking or recombinant protein controls to confirm binding specificity to the intended target .

How can researchers distinguish between specific and non-specific binding in RPS13 immunostaining?

Distinguishing specific from non-specific binding in RPS13 immunostaining requires implementation of rigorous controls and validation strategies. Researchers should first perform titration experiments to determine the minimal antibody concentration that provides clear signal above background. Pre-absorption controls, where the antibody is pre-incubated with excess purified RPS13 protein or immunizing peptide before application to samples, can help identify specific binding. For genetic validation, siRNA knockdown or CRISPR knockout of RPS13 in cell lines should substantially reduce or eliminate specific immunostaining. Secondary antibody-only controls are essential to identify background signal. When troubleshooting high background, researchers should optimize blocking conditions (typically 3-5% BSA or normal serum from the secondary antibody host species) and consider adding 0.1-0.3% Triton X-100 for intracellular proteins like RPS13. For immunofluorescence applications, HeLa cells have been validated as positive controls. Additionally, comparison of staining patterns using multiple antibodies targeting different epitopes of RPS13 can provide further confidence in signal specificity. Researchers should also be aware that RPS13's cytoplasmic localization pattern should be consistent with ribosomal distribution, providing another criterion for distinguishing specific from non-specific signals .

How can RPS13 antibodies be effectively used in cancer research applications?

RPS13 antibodies offer valuable tools for cancer research applications, particularly given emerging evidence of ribosomal protein involvement in tumorigenesis. For immunohistochemical analysis of tumor tissues, RPS13 antibodies have been successfully applied to human colon cancer samples using recommended dilutions of 1:50-1:500 with TE buffer (pH 9.0) for antigen retrieval. Researchers investigating potential differential expression of RPS13 in tumors versus normal tissues should implement matched normal-tumor pairs with standardized staining protocols and quantification methods. Co-immunoprecipitation experiments using RPS13 antibodies can identify novel interaction partners that may be dysregulated in cancer contexts. For analyzing potential associations between RPS13 expression and clinical outcomes, tissue microarray approaches coupled with digital pathology quantification allow high-throughput analysis across large patient cohorts. Research has indicated potential roles for RPS13 in multidrug-resistant gastric cancer cells, suggesting utility in studying chemoresistance mechanisms. When preparing monoclonal antibodies against RPS13 for cancer research, prokaryotic expression systems have successfully generated immunogenic His-RPS13 fusion proteins that induce high-titer antibody responses in immunized mice. These antibodies can subsequently be used to investigate RPS13's specific functions in tumorigenesis, potentially revealing novel therapeutic targets or biomarkers in cancer progression .

What controls should be included when using RPS13 antibodies in experimental procedures?

Implementing comprehensive controls when using RPS13 antibodies is essential for experimental validity and reproducibility. Positive controls should include samples with confirmed RPS13 expression, such as MCF-7 cells for Western blot or HeLa cells for immunofluorescence applications. Negative controls should incorporate secondary antibody-only treatments to identify non-specific binding of detection reagents. For genetic validation, siRNA knockdown or CRISPR knockout of RPS13 provides the most stringent control for antibody specificity, though researchers should be aware that complete RPS13 elimination may not be compatible with cell viability due to its essential role in ribosome function. Peptide competition assays, where excess immunizing peptide blocks specific antibody binding, offer another approach to demonstrate specificity. Loading controls for Western blot should be selected based on experimental context—total protein stains (Ponceau S or Amido Black) often provide more reliable normalization than individual housekeeping proteins across diverse experimental conditions. When comparing RPS13 expression across tissue or cell types, researchers should include multiple independent biological replicates to account for natural variation and include taxonomically diverse samples when validating antibodies marketed for cross-species reactivity. Technical replicates should be included to assess methodological reproducibility, and quantification methods should be established prior to data collection to prevent post-hoc adjustments that might introduce bias .

How should researchers approach epitope mapping and selection for custom RPS13 antibody production?

When approaching epitope mapping and selection for custom RPS13 antibody production, researchers should carefully consider several structural and sequence-based factors. Bioinformatic analysis should first identify regions of RPS13 with high antigenicity scores using algorithms like Kyte-Doolittle or Hopp-Woods scales while avoiding regions with high sequence conservation with other ribosomal proteins to minimize cross-reactivity. The middle region of human RPS13 (amino acids 79-151) has been successfully used as an immunogen, particularly the sequence "ILRILKSKGL APDLPEDLYH LIKKAVAVRK HLERNRKDKD AKFRLILIES." Researchers should preferentially select epitopes predicted to be surface-exposed in the native protein conformation, as determined through structural modeling or crystallographic data. For producing antibodies with cross-species reactivity, epitopes should be selected from regions with high sequence conservation across target species—analysis indicates 100% identity across many mammalian species in key regions of RPS13. Conversely, for species-specific antibodies, unique sequence regions should be identified. When synthesizing peptide antigens, N-terminal or C-terminal cysteine residues can be added for conjugation to carrier proteins like KLH or BSA to enhance immunogenicity. For recombinant protein immunogens, prokaryotic expression in E. coli BL21 using vectors like pET-28a(+) has been successfully employed to produce His-tagged RPS13 that can be purified via affinity chromatography and confirmed by SDS-PAGE and Western blot before immunization. Complete Freund's adjuvant for primary immunization followed by incomplete Freund's adjuvant for boosters has proven effective for generating high-titer antibody responses in rabbits and mice .

What methodological approaches can resolve contradictory results when using different RPS13 antibodies?

Resolving contradictory results when using different RPS13 antibodies requires systematic methodological investigation across multiple dimensions. Researchers should first determine whether discrepancies stem from fundamental differences in epitope recognition by comparing the immunogens used to generate each antibody—antibodies targeting different domains of RPS13 may legitimately yield different results if post-translational modifications, protein interactions, or conformational changes differentially affect epitope accessibility. Cross-validation with orthogonal techniques is essential—if antibodies show discrepancies in immunostaining patterns, researchers should employ Western blot, mass spectrometry, or RNA expression analysis to establish baseline expression levels and expected distribution patterns. Antibody validation using genetic approaches provides particularly strong evidence—comparing antibody performance in wild-type versus RPS13 knockdown/knockout samples can definitively establish specificity. For antibodies yielding similar qualitative but different quantitative results, standardization of protocols including sample preparation, antibody concentration, incubation conditions, and detection methods may resolve apparent contradictions. When differences persist despite standardization, it may indicate that antibodies recognize distinct pools or modified forms of RPS13. Researchers should consider performing immunoprecipitation followed by mass spectrometry to identify precisely what each antibody is detecting. Additionally, the use of tagged RPS13 constructs (such as FLAG or GFP fusions) allows comparison between antibody-based detection and tag-based detection to resolve discrepancies. Finally, rigorous peer review of contradictory results, including consultation with antibody manufacturers regarding batch variations or known limitations, may provide important context for interpreting apparently conflicting observations .

How can RPS13 antibodies be applied in ribosomal biogenesis and function studies?

RPS13 antibodies offer powerful tools for investigating ribosomal biogenesis and function through multiple experimental approaches. For studying ribosomal assembly dynamics, researchers can employ RPS13 antibodies in pulse-chase experiments combined with immunoprecipitation to track incorporation of newly synthesized RPS13 into ribosomal subunits. Polysome profiling coupled with Western blot analysis using RPS13 antibodies can assess distribution of this protein across monosomes, polysomes, and free subunits under various cellular conditions like stress or differentiation. Chromatin immunoprecipitation (ChIP) using antibodies against transcription factors combined with RPS13 expression analysis can elucidate regulatory mechanisms controlling ribosomal protein synthesis. For spatial organization studies, super-resolution microscopy techniques such as STORM or PALM using fluorophore-conjugated RPS13 antibodies can reveal nanoscale distribution of ribosomes within cellular compartments. Co-immunoprecipitation experiments with RPS13 antibodies can identify novel interaction partners involved in ribosome assembly or extraribosomal functions. In tissue-specific ribosome studies, RPS13 antibodies can help characterize potential heterogeneity in ribosome composition across different cell types. For translational regulation research, proximity ligation assays combining RPS13 antibodies with antibodies against translation factors or mRNA-binding proteins can reveal spatial relationships during active translation. Finally, RPS13 antibodies can support investigation of ribosomopathies by comparing expression, localization, and post-translational modifications of RPS13 in patient-derived versus healthy control samples .

What are the emerging applications of RPS13 antibodies in cancer biology research?

Emerging applications of RPS13 antibodies in cancer biology research span diagnostic, mechanistic, and therapeutic dimensions. For diagnostic applications, immunohistochemical analysis of tissue microarrays using standardized RPS13 staining protocols can identify potential associations between expression levels and clinical outcomes across cancer types. Mechanistically, co-immunoprecipitation using RPS13 antibodies followed by mass spectrometry can identify cancer-specific interaction partners that might represent novel therapeutic targets. Research has indicated potential roles for RPS13 in multidrug-resistant gastric cancer cells, suggesting utility in studying chemoresistance mechanisms through comparative expression analysis between sensitive and resistant cell lines. Combined RNA-seq and RPS13 immunoprecipitation followed by RNA sequencing (RIP-seq) can identify cancer-specific mRNAs preferentially translated by ribosomes containing RPS13, potentially revealing specialized translational programs in malignancy. CRISPR screens examining synthetic lethality with RPS13 modulation could identify novel therapeutic vulnerabilities in cancer cells with altered ribosomal biology. For investigating extraribosomal functions, proximity-dependent biotinylation (BioID) with RPS13 fusion proteins combined with antibody-based validation can map cancer-specific RPS13 interaction networks. Dual immunofluorescence with RPS13 antibodies and markers of stress granules or P-bodies can investigate potential roles in stress response pathways frequently dysregulated in cancer. Finally, circulating tumor cell analysis using RPS13 antibodies in combination with epithelial markers might offer novel diagnostic approaches, particularly if cancer-specific RPS13 modifications are identified that can be detected with specialized antibodies .

How can post-translational modifications of RPS13 be detected and characterized using specialized antibodies?

Detection and characterization of post-translational modifications (PTMs) on RPS13 require specialized antibody approaches and complementary techniques. Researchers investigating RPS13 phosphorylation should develop or acquire phospho-specific antibodies targeting known or predicted phosphorylation sites, validating specificity through lambda phosphatase treatment of samples or comparison with phospho-deficient mutants (e.g., S→A substitutions). For ubiquitination studies, immunoprecipitation with RPS13 antibodies followed by Western blotting with anti-ubiquitin antibodies can identify modified forms, while the reverse approach (ubiquitin IP followed by RPS13 detection) can confirm findings. SUMOylation can be approached similarly, using SUMO-specific antibodies. Mass spectrometry remains the gold standard for comprehensive PTM mapping—immunoprecipitation with RPS13 antibodies followed by MS/MS analysis can identify sites of modification and relative abundances. For acetylation studies, pan-acetyl-lysine antibodies can be used following RPS13 immunoprecipitation, with site-specific acetylation antibodies developed for confirmed sites of interest. Temporal dynamics of modifications can be studied through pulse-chase experiments combined with modification-specific antibodies. Subcellular fractionation followed by Western blotting with modification-specific antibodies can reveal compartment-specific patterns of RPS13 modification. For functional studies, comparing wild-type and modification-site mutant RPS13 can elucidate roles of specific PTMs, with antibodies used to assess incorporation into ribosomes or interaction with regulatory partners. Finally, developing antibodies that specifically recognize conformational changes induced by PTMs represents a frontier application that could reveal how modifications alter RPS13's structural properties and functional interactions within the translational machinery .