RPSA Antibody

What is RPSA and why is it a significant target for antibody development?

RPSA (Ribosomal Protein SA) is a multifunctional protein that serves dual roles in cellular biology. It functions as a component of the 40S ribosomal subunit, where it's required for assembly and stability, and also acts as a cell surface receptor for laminin (hence its alternative names 67kDa Laminin Receptor and 37kDa Laminin Receptor Precursor). RPSA plays critical roles in ribosomal RNA maturation, cell adhesion to basement membranes, and signal transduction pathway activation. It's also implicated in cell fate determination and tissue morphogenesis . Additionally, RPSA serves as a receptor for various pathogens including viruses (Adeno-associated viruses, Dengue virus, Sindbis virus), prions, and certain bacteria, making it significant in infectious disease research .

What are the standard applications for RPSA antibodies in research?

RPSA antibodies are employed across multiple experimental techniques:

What are the common forms of RPSA detected by antibodies?

Researchers should be aware that RPSA exists in multiple molecular forms:

• The monomeric form at approximately 37 kDa (37LRP)

• A ~53 kDa form referred to as 53LR

• The 67 kDa laminin receptor (67LR)

The higher molecular weight forms are thought to result from post-translational modifications, including potential SUMOylation. While 37LRP is detected in both cytosolic and membrane fractions, 67LR is typically detected exclusively in membrane fractions . These different forms may have distinct biological functions, and antibody selection should consider which form(s) are relevant to the research question.

How do I determine the most appropriate RPSA antibody clonality for my experiment?

The choice between monoclonal and polyclonal RPSA antibodies depends on your experimental goals:

For detecting specific post-translational modifications or particular molecular weight forms of RPSA, epitope-specific antibodies targeting the relevant regions should be selected .

What validation criteria should I use when selecting an RPSA antibody?

Based on established validation criteria for antibodies used in RPPA studies, which are applicable to other applications, consider the following:

• Specificity validation: The antibody should demonstrate a single or dominant band in Western blot around the expected molecular weight (37-40 kDa for monomeric RPSA) .

• Quantitative validation: Good correlation (Pearson >0.7) between abundances measured by the primary detection method and a secondary validation method (e.g., RPPA vs. Western blot) .

• Cross-reactivity testing: Confirm the antibody's reactivity with your species of interest. Many RPSA antibodies are reactive with human, mouse, and rat samples, but this varies by product .

• Application-specific validation: Test the antibody in your specific application before proceeding with critical experiments .

• Knockout/knockdown validation: Use RPSA knockdown or knockout samples as negative controls to confirm specificity .

How do I distinguish between different molecular weight forms of RPSA in my experiments?

To effectively distinguish between the different molecular weight forms of RPSA (37LRP, 53LR, and 67LR):

• Use gradient gels (e.g., 4-15%) for better separation of different molecular weight species.

• Include appropriate subcellular fractionation - 67LR is reported to be exclusively in membrane fractions while 37LRP is present in both cytosolic and membrane fractions .

• Consider siRNA experiments targeting the SUMO pathway. Research indicates that knockdown of UBC9/UBE2I (the SUMO E2 conjugating enzyme) caused a marked decrease in higher molecular weight RPSA species, particularly 53LR, suggesting these forms may be SUMOylated variants .

• Be aware that detection of 67LR may be condition-sensitive, making it inconsistently observed across experiments .

• To confirm identity of bands, use mass spectrometry analysis of immunoprecipitated proteins from the appropriate molecular weight ranges.

What factors affect the reliability of RPSA antibody measurements in multi-platform studies?

Several factors influence the reliability of RPSA measurements across different platforms:

• Antibody validation status: In RPPA studies, antibodies classified as "Valid" showed significantly higher correlations with mRNA expression levels compared to those labeled "Use with Caution" .

• Platform compatibility: When comparing RPPA and mass spectrometry measurements of the same proteins across cell lines, proteins detected with more reliable antibodies demonstrated higher correlation between the two measurement techniques .

• Post-translational modifications: Different antibodies may have varying affinities for modified forms of RPSA, affecting quantification results .

• Epitope accessibility: The subcellular localization and protein-protein interactions of RPSA may mask epitopes in certain experimental conditions .

• Assay conditions: Optimization of incubation conditions for individual antibodies can significantly improve reliability but may not be possible in high-throughput platforms like RPPA .

To account for these factors, researchers should consider using multiple antibodies targeting different epitopes or combining antibody-based detection with orthogonal methods like mass spectrometry for critical measurements .

Why might my RPSA antibody detect unexpected molecular weight species?

Detection of unexpected molecular weight species when using RPSA antibodies may occur for several reasons:

• Post-translational modifications: SUMOylation of RPSA has been detected in multiple proteomics screens, which can increase the apparent molecular weight. Knockdown of the SUMOylation pathway (UBC9/UBE2I) reduced the detection of the 53 kDa form of RPSA .

• Alternative splicing or proteolytic processing: The transition between the 37 kDa and 67 kDa forms is not fully understood and may involve mechanisms beyond simple dimerization .

• Protein-protein interactions: Stable interactions with other proteins may result in incomplete denaturation during sample preparation.

• Non-specific binding: Some antibodies may cross-react with structurally similar proteins. This is particularly problematic in techniques like RPPA where molecular weight separation is not possible .

• Epitope tagging effects: Research has shown that FLAG-tagged RPSA constructs failed to form the higher molecular weight species (53LR, 67LR) despite their presence when detected with anti-RPSA antibodies, suggesting the tag may interfere with natural processing or detection .

For accurate interpretation, validation experiments including knockdown/knockout controls and comparison with multiple antibodies targeting different epitopes are recommended .

How can I improve the specificity of RPSA detection in immunohistochemistry applications?

To enhance specificity in IHC applications:

• Optimize antigen retrieval: Different RPSA antibodies may require specific retrieval methods. For example, some products suggest TE buffer pH 9.0 as the primary method, with citrate buffer pH 6.0 as an alternative .

• Titrate antibody concentration: The recommended dilution ranges for IHC with RPSA antibodies vary widely (1:50-1:2000), indicating the need for empirical optimization for each tissue type and fixation method .

• Include appropriate controls:

  • Positive control tissues known to express RPSA (e.g., lung cancer tissue, tonsil, or cervical carcinoma)

  • Negative controls omitting primary antibody

  • Ideally, RPSA-depleted tissues or cells as specificity controls

• Consider signal amplification methods for detecting lower expression levels while maintaining specificity.

• For multiplex staining, test for potential cross-reactivity between antibodies in your panel.

• Compare results with orthogonal methods (e.g., in situ hybridization for mRNA expression) to validate findings in critical studies .

How do I interpret discrepancies between mRNA and protein levels when studying RPSA?

Discrepancies between RPSA mRNA and protein levels should be interpreted considering several factors:

• Antibody reliability significantly impacts observed mRNA-protein correlations. Research shows that proteins measured with less reliable antibodies have lower observed mRNA-protein correlations in RPPA studies .

• The same discrepancy is not observed when RPSA is measured using mass spectrometry, suggesting the divergence may be technical rather than biological .

• Post-translational regulation: RPSA undergoes complex post-translational processing, including potential SUMOylation and the formation of higher molecular weight species (53LR, 67LR) .

• Subcellular localization: RPSA distributes across multiple cellular compartments (cytosol, ribosomes, membrane, cytoskeleton, and nucleus), and some antibodies may preferentially detect specific pools .

• Tissue/cell-specific regulation: The correlation between RPSA mRNA and protein may vary across different tissues or cellular states.

To address these challenges, researchers should:

• Use multiple antibodies targeting different epitopes

• Compare results from different detection methods (e.g., RPPA vs. mass spectrometry)

• Include appropriate subcellular fractionation in analyses

• Consider the validation status of antibodies when interpreting correlation data

What considerations are important when comparing RPSA detection across different experimental platforms?

When comparing RPSA measurements across different platforms:

• Antibody consistency: Even when targeting the same protein, different antibodies can yield varying results. In multi-platform RPPA studies, antibodies targeting the same antigen clustered together in hierarchical clustering analyses, but the degree of correlation varied between platforms .

• Platform-specific artifacts: Each technique (WB, IHC, IF, RPPA, mass spectrometry) has inherent biases that affect RPSA quantification:

  • Western blotting allows molecular weight discrimination but has limited quantitative range

  • RPPA provides better quantification but lacks molecular weight discrimination

  • Mass spectrometry offers unbiased detection but may miss specific post-translational modifications

• Data normalization: Different normalization methods can significantly impact the correlation between platforms. Research indicates that correlations between normalized RPPA data from different antibodies were lower than correlations between raw data, resulting in better separation of truly concordant measurements .

• Epitope accessibility: Sample preparation for different techniques may expose or mask specific epitopes, affecting detection.

What emerging technologies are improving RPSA antibody reliability and application range?

Several technological advances are enhancing RPSA antibody applications:

• Recombinant antibody technology: The development of recombinant RPSA antibodies (e.g., 83495-5-PBS) provides unrivaled batch-to-batch consistency, easy scale-up, and better security of supply compared to traditional hybridoma-produced antibodies .

• Validation databases: Increased sharing of antibody validation data through repositories and literature allows researchers to make more informed selections based on application-specific performance.

• Antibody engineering: Site-specific conjugation strategies enable better control over antibody labeling, improving performance in multiplexed applications.

• Orthogonal validation approaches: Integration of antibody-based detection with mass spectrometry or genomic approaches provides more robust validation frameworks.

• Advanced antibody pairs: Development of matched antibody pairs specifically designed for quantitative assays like sandwich ELISA or cytometric bead arrays enhances sensitivity and specificity .

These advances will continue to improve the reliability and expand the application range of RPSA antibodies in research and diagnostic settings.

How might RPSA antibodies contribute to understanding disease mechanisms beyond cancer?

While much RPSA research focuses on cancer due to its overexpression in various cancer cell lines , RPSA antibodies have potential applications in studying:

• Infectious disease: RPSA acts as a receptor for multiple pathogens including prions, viruses (Adeno-associated viruses, Dengue virus, Sindbis virus, Venezuelan equine encephalitis virus), and bacteria . RPSA antibodies could help elucidate host-pathogen interactions and identify potential therapeutic targets.

• Developmental biology: RPSA may play roles in cell fate determination and tissue morphogenesis . Antibodies could track expression patterns during development.

• Asplenia: The RPSA gene has been associated with the disease Asplenia . Antibodies may help characterize the molecular mechanisms underlying this condition.

• Neurodegenerative diseases: Given RPSA's role as a receptor for pathogenic prion proteins , antibodies could provide insights into prion disease mechanisms.

• Prokaryotic research: Bacterial RPSA (30S ribosomal protein S1) antibodies are available for studying prokaryotic translation systems, potentially contributing to antibiotic development .

As research expands beyond cancer, RPSA antibodies will likely become valuable tools for exploring these diverse pathological and physiological processes.