RPS24 Antibody

What is RPS24 and why is it significant in molecular research?

RPS24 (Ribosomal Protein S24) is a crucial component of the ribosome that plays a key role in the translation of mRNA into proteins. Its significance extends beyond basic protein synthesis, as it's involved in 18S rRNA maturation and has been implicated in various pathological conditions. Recent research has shown that RPS24 overexpression contributes to unfavorable prognosis in hepatocellular carcinoma (HCC) patients by facilitating cell proliferation and creating an immunosuppressive microenvironment . In non-cancerous contexts, RPS24 functions in the ITS1-first pathway of ribosomal RNA processing and may participate in transcriptional repression of 45S rDNA . The protein's involvement in these diverse cellular processes makes it a valuable target for molecular and biomedical research.

What are the common applications for RPS24 antibodies in research settings?

RPS24 antibodies are versatile research tools with validated applications including:

These applications enable researchers to detect, quantify, and visualize RPS24 expression across various experimental systems, making them invaluable for studying ribosomal biology, cancer mechanisms, and cellular stress responses .

How should researchers optimize RPS24 antibody conditions for Western blot applications?

For optimal Western blot results with RPS24 antibodies, researchers should consider the following methodological approach:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for efficient extraction of RPS24 from cellular compartments.

• Loading control selection: Consider using GAPDH or β-actin as loading controls, with careful normalization since ribosomal protein expression can affect global translation.

• Protein loading: Start with 20-30μg of total protein lysate per lane.

• Antibody dilution: Begin with the manufacturer's recommended dilution (typically 1:500 for RPS24 antibodies) and optimize as needed.

• Incubation conditions: Perform primary antibody incubation overnight at 4°C to maximize specific binding.

• Detection method selection: HRP-conjugated secondary antibodies with ECL detection systems have proven effective for RPS24 visualization.

• Expected molecular weight: Confirmed RPS24 bands should appear at approximately 15 kDa.

Validation studies have demonstrated positive Western blot detection in multiple cell types including SH-SY5Y, HEK-293, and HL-60 cells , providing reliable positive controls for experimental setup.

What considerations are important when using RPS24 antibodies for immunohistochemistry?

When conducting immunohistochemistry studies with RPS24 antibodies, researchers should implement the following methodological guidelines:

• Fixation protocol: Formalin-fixed, paraffin-embedded (FFPE) tissues are compatible with RPS24 detection.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended for optimal antigen exposure.

• Blocking strategy: Use 5-10% normal serum from the same species as the secondary antibody for 1 hour at room temperature.

• Antibody dilution: Begin with 1:50-1:200 dilution in antibody diluent buffer, optimizing as needed.

• Incubation parameters: Overnight incubation at 4°C typically yields optimal results.

• Controls: Include both negative controls (omitting primary antibody) and positive tissue controls (mouse liver, mouse pancreas, rat liver) for validation.

• Counterstaining: Hematoxylin counterstaining provides good nuclear contrast against cytoplasmic RPS24 staining.

For monitoring tumor proliferation in xenograft models, RPS24 immunohistochemistry can be complemented with Ki67 staining, as demonstrated in studies of RPS24 knockdown effects on HCC tumor growth .

How can researchers distinguish between RPS24 isoforms using antibody-based techniques?

Distinguishing between RPS24 isoforms (particularly RPS24L and RPS24S) requires strategic experimental design:

• Antibody selection: Choose antibodies targeting regions that differ between isoforms or use paired antibodies (one common epitope, one isoform-specific).

• Western blot resolution: Use 15% polyacrylamide gels with extended run times to resolve the small size difference between isoforms.

• Pre-analytical preparation: Consider phosphatase treatment of samples, as differential phosphorylation may affect mobility.

• Isoform-specific controls: Generate overexpression constructs of individual isoforms as positive controls.

• Validation approach: Confirm specificity using cells treated with siRNAs targeting specific isoforms.

Research has shown that different cell lines display varying baseline ratios of RPS24L/S , making it essential to establish baseline patterns in your experimental system before investigating changes under different conditions. For hypoxia studies, include timepoints between 0-24 hours, as hypoxia increases the relative abundance of RPS24L through autophagy-dependent mechanisms .

How can RPS24 antibodies be employed to study the relationship between RPS24 and cancer progression?

To investigate RPS24's role in cancer progression using antibody-based techniques, researchers should consider the following methodological framework:

• Expression correlation studies: Use RPS24 immunohistochemistry on tissue microarrays containing tumor and adjacent normal tissues, scoring expression levels and correlating with clinical parameters.

• Survival analysis: Stratify patients based on RPS24 staining intensity and perform Kaplan-Meier analysis to assess prognostic value.

• Mechanistic investigation: Combine RPS24 immunoblotting with cell proliferation assays (CCK-8, colony formation) following RPS24 knockdown or overexpression.

• In vivo models: Establish xenograft models using cells with modulated RPS24 expression and evaluate tumor growth parameters and RPS24/Ki67 immunostaining.

• Pathway analysis: Correlate RPS24 expression with markers of cell cycle progression (E2F targets, G2M checkpoint proteins) through co-immunoprecipitation or co-immunostaining approaches.

What experimental approaches can assess the relationship between RPS24 and immune system functioning?

To explore RPS24's impact on immune responses, particularly in the context of cancer immunotherapy, researchers should consider these methodological approaches:

• Immune infiltration analysis: Perform multiplex immunofluorescence with RPS24 antibodies alongside markers for specific immune cell populations (DCs, T helper cells, Tcm, Th17 cells).

• Correlation with immune checkpoints: Use co-immunostaining or sequential immunoblotting to evaluate relationships between RPS24 and immune checkpoint molecules (CTLA4, HAVCR2, LAG3, PDCD1).

• Functional assessment: In co-culture systems, evaluate immune cell functionality (cytokine production, proliferation) when exposed to cancer cells with different RPS24 expression levels.

• Immunotherapy response prediction: Stratify patients by RPS24 expression and correlate with TIDE scores or actual immunotherapy responses.

Research has demonstrated that RPS24 expression negatively correlates with infiltration levels of several immune cell types, including dendritic cells and various T helper cell populations . Furthermore, genes associated with immune checkpoints (CTLA4, HAVCR2, LAG3, PDCD1) show significant increases in high-RPS24-expression groups, suggesting RPS24 may enhance immune evasion mechanisms in cancer .

How can researchers investigate RPS24's role in ribosomal RNA processing?

To study RPS24's function in rRNA processing, implement the following methodological approaches:

• Pre-rRNA processing analysis: Use Northern blotting with probes targeting different regions of the pre-rRNA transcript to detect processing intermediates.

• Co-immunoprecipitation studies: Employ RPS24 antibodies for immunoprecipitation followed by RNA extraction and RT-qPCR to identify associated rRNA species.

• Subcellular localization: Perform immunofluorescence co-staining with RPS24 antibodies and nucleolar markers like fibrillarin to assess nucleolar localization and morphology.

• Genetic perturbation: Analyze pre-rRNA profiles using a bioanalyzer following RPS24 knockdown or mutation.

• Ribosome profiling: Combine with RPS24 immunoprecipitation to assess the impact on mature ribosome composition.

Studies have shown that impaired RPS24 function leads to accumulation of specific pre-rRNA species and reduced mature 18S rRNA levels. In Arabidopsis models, mutations affecting RPS24 function resulted in a 4% reduction in the 18S/25S rRNA ratio , while in human cells, RPS24 deficiency caused a 40% reduction in mature 18S rRNA levels, with specific reductions in 21S and 18S-E precursors .

What methods can be used to study RPS24 function under cellular stress conditions?

To investigate RPS24's role during cellular stress, particularly hypoxia, researchers should consider these methodological approaches:

• Isoform-specific detection: Use Western blotting optimized to distinguish between RPS24L and RPS24S isoforms under varying oxygen conditions.

• Pathway inhibition studies: Couple RPS24 antibody detection with inhibitors of autophagy (3-MA, chloroquine) or mTORC1 (rapamycin) to dissect regulatory mechanisms.

• Temporal analysis: Conduct time-course studies of RPS24 isoform shifts during hypoxia induction and recovery.

• 3D culture models: Implement spheroid or organoid cultures with immunostaining for RPS24 to assess expression in physiologically relevant oxygen gradients.

• Stability assessment: Perform cycloheximide chase experiments with RPS24 antibody detection to compare protein stability between isoforms.

Research has revealed that hypoxic conditions increase the relative abundance of RPS24L compared to RPS24S through differential regulation: RPS24L is induced in an autophagy-dependent manner, while RPS24S is reduced via mTORC1 repression in a HIF-dependent manner . The longer isoform (RPS24L) produces a more stable protein that enhances cell survival under hypoxic conditions .

What are the common challenges in RPS24 antibody applications and how can they be addressed?

Researchers frequently encounter these challenges when working with RPS24 antibodies:

For specific applications like immunoprecipitation, using 0.5-4μg antibody for 200-400μg of whole cell extracts has been validated as effective . When performing Western blot analysis, the expected molecular weight of approximately 15 kDa should be confirmed against appropriate positive controls such as U-87MG, 293T, HeLa, or Jurkat cell lysates .

How should researchers validate the specificity of RPS24 antibodies?

To ensure ribosomal protein S24 antibody specificity, implement this comprehensive validation strategy:

• Positive control selection: Include lysates from cells known to express RPS24 (SH-SY5Y, HEK-293, HL-60) .

• Negative control generation: Use RPS24 knockdown or knockout samples (via siRNA or CRISPR).

• Peptide competition: Pre-incubate antibody with excess immunizing peptide before application.

• Cross-reactivity assessment: Test the antibody against recombinant RPS24 and closely related ribosomal proteins.

• Multi-technique confirmation: Verify consistent results across different detection methods (WB, IHC, IF).

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm capture of authentic RPS24.

• Orthogonal detection methods: Compare protein detection with mRNA expression by RT-qPCR.

This comprehensive validation approach is particularly important given RPS24's involvement in multiple cellular processes and presence of different isoforms that may be differentially regulated in various physiological conditions .

How might RPS24 antibodies contribute to developing novel cancer therapies?

RPS24 antibodies can facilitate the development of targeted cancer therapies through several strategic research approaches:

• Biomarker development: Use RPS24 antibodies to stratify patient tumor samples based on expression levels, facilitating the identification of patients who might benefit from specific therapeutic approaches.

• Therapeutic target validation: Employ RPS24 antibodies in target validation studies to confirm the role of RPS24 in cancer progression and therapy resistance mechanisms.

• Mechanism exploration: Utilize antibody-based techniques to uncover downstream effectors and interaction partners that could serve as alternative therapeutic targets.

• Immunotherapy enhancement: Investigate how modulation of RPS24 affects immune checkpoint expression and immune cell infiltration, potentially revealing strategies to enhance immunotherapy efficacy.

• Resistance mechanism identification: Apply RPS24 antibodies to comparative studies of treatment-naïve versus resistant tumors to understand the protein's role in therapy resistance.

Research has already demonstrated that RPS24 knockdown significantly inhibits the proliferation of HCC cells and reduces tumor growth in xenograft models . Additionally, the correlation between RPS24 expression and immune checkpoint molecules suggests targeting RPS24 might enhance responses to immunotherapy by altering the immunosuppressive tumor microenvironment .

What emerging methodologies might enhance RPS24 antibody applications in future research?

Emerging technologies promise to expand the utility of RPS24 antibodies in molecular research:

• Spatial transcriptomics integration: Combine RPS24 immunostaining with spatial transcriptomics to correlate protein expression with local gene expression profiles in tumor microenvironments.

• Single-cell proteomics: Apply RPS24 antibodies in single-cell Western blot or mass cytometry approaches to examine cell-to-cell variability in expression.

• Live-cell imaging: Develop non-perturbing antibody fragments or nanobodies against RPS24 for real-time visualization of its dynamics during stress responses.

• Proximity labeling: Use RPS24 antibodies in conjunction with proximity ligation assays to map context-specific protein interaction networks.

• Antibody engineering: Create bispecific antibodies targeting RPS24 and other markers to enhance detection sensitivity in specific cellular compartments.

These approaches would be particularly valuable for investigating the differential regulation of RPS24 isoforms under various stress conditions, such as the autophagy-dependent induction of RPS24L under hypoxia , and could reveal new therapeutic vulnerabilities in cancer and other diseases.