RPS26 Antibody

What is RPS26 and why is it significant in molecular biology research?

RPS26 (ribosomal protein S26) is an essential component of the eukaryotic 40S ribosomal subunit, located near the mRNA exit site region. With a calculated molecular weight of 13 kDa (though typically observed at 18-21 kDa in Western blots), this 115-amino acid protein plays critical roles in ribosome biogenesis and protein synthesis .

Research significance stems from several areas: First, mutations in RPS26 are associated with Diamond-Blackfan anemia (DBA10), a congenital red cell aplasia usually diagnosed in early infancy . Second, RPS26 significantly influences 80S ribosome assembly, with studies showing that mutations in the protein can lead to altered ratios of 40S/60S ribosomal subunits . Finally, RPS26 has been implicated in regulating p53 activity, nonsense-mediated mRNA decay, and mRNA-specific translation through Kozak sequence recognition .

What applications are RPS26 antibodies validated for?

RPS26 antibodies are validated for multiple research applications with specific recommended parameters:

While these applications represent validated uses, the actual working concentration should be optimized for specific experimental conditions .

What is known about RPS26 structure and its eukaryote-specific motif?

RPS26 contains a eukaryote-specific motif within the Y62–K70 segment that has been the subject of significant research. Experimental evidence indicates that this motif is crucial for proper protein function, as:

• Complete deletion of the Y62–K70 segment is lethal in yeast models

• Simultaneous replacement of five conserved residues within this segment by alanines results in growth defects under stress conditions

• These mutations produce distinct changes in polysome profiles, indicating accumulation of free 60S subunits

How should researchers optimize Western blot protocols for RPS26 detection?

For optimal Western blot detection of RPS26, researchers should consider the following protocol modifications:

• Sample preparation: Use RIPA buffer with protease inhibitors for efficient extraction of ribosomal proteins

• Gel percentage: Utilize 15% SDS-PAGE gels for better resolution of the 18-21 kDa RPS26 protein

• Transfer conditions: Extend transfer time for small proteins or consider semi-dry transfer systems

• Antibody dilution: Start with 1:1000 dilution and adjust based on signal strength

• Signal detection: Use ECL Plus or similar enhanced chemiluminescence systems for sensitive detection

When troubleshooting, note that RPS26 consistently appears at 18-21 kDa despite its calculated molecular weight of 13 kDa . This discrepancy is likely due to post-translational modifications or the protein's highly basic nature. Always include positive control samples such as mouse ovary tissue, mouse lung tissue, or cell lines like 293T, HEL, or MCF-7 .

What controls are essential when working with RPS26 antibodies?

Implementing appropriate controls is critical for reliable interpretation of results with RPS26 antibodies:

Positive controls:

• Western blot: Mouse ovary/lung tissue, human cell lines (293T, HEL, Caco-2, PC-3, MCF-7)

• IHC: Human breast cancer tissue, human kidney tissue

• IF/ICC: MCF-7 cells

Negative controls:

• Primary antibody omission control: Replace RPS26 antibody with diluent alone

• Isotype control: Use non-specific rabbit IgG at equivalent concentration

• Genetic approach: RPS26 knockdown samples (complete knockout may be lethal)

Additional validation approaches:

• Multiple antibody concordance: Confirm findings using antibodies against different RPS26 epitopes

• Western blot correlation: Validate IHC/IF findings with corresponding Western blot analysis

• Sample panel validation: Test across multiple tissues/cell types with known expression patterns

How can researchers validate RPS26 antibody specificity?

Antibody validation is essential for generating reliable data. For RPS26 antibodies, consider these validation strategies:

• Western blot analysis:

  • Verify correct molecular weight (18-21 kDa)

  • Observe a single predominant band in multiple cell/tissue types

  • Compare band intensity across samples with expected expression levels

• Genetic approaches:

  • Perform siRNA knockdown of RPS26 (note that complete knockdown may be lethal)

  • Compare antibody signal before and after knockdown

• Immunoprecipitation validation:

  • Perform IP followed by Western blot detection with a different RPS26 antibody

  • Analyze IP products by mass spectrometry to confirm RPS26 identity

• Cross-application validation:

  • Confirm that samples positive by Western blot also show positivity in IHC/IF

  • Compare subcellular localization patterns across multiple detection methods

How can RPS26 antibodies be used to study Diamond-Blackfan anemia?

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia associated with mutations in several ribosomal protein genes, including RPS26 (causing DBA10) . RPS26 antibodies provide valuable tools for studying the molecular mechanisms of this disease:

• Expression analysis:

  • Compare RPS26 protein levels between patient-derived cells and healthy controls

  • Assess whether specific mutations affect protein stability or expression

  • Correlate expression levels with disease severity or treatment response

• Mutation effect characterization:

  • Use antibodies recognizing different epitopes to determine if mutations affect antibody binding

  • For frameshift mutations like c.96dupG (p.K32fsX5), antibodies targeting N-terminal vs. C-terminal regions can confirm truncation

• Ribosome assembly analysis:

  • Perform sucrose gradient fractionation followed by Western blot to examine incorporation of RPS26 into 40S subunits

  • Compare ribosome profiles between patient samples and controls to identify assembly defects

• Erythroid differentiation models:

  • Track RPS26 expression during normal and abnormal erythropoiesis

  • Correlate with erythroid maturation markers to identify stage-specific defects

  • Assess ribosome function in translating erythroid-specific mRNAs

What experimental approaches can assess RPS26's role in ribosome assembly?

RPS26 plays a critical role in the assembly of the 40S ribosomal subunit. Several experimental approaches using RPS26 antibodies can assess this function:

• Polysome profiling:

  • Sucrose gradient ultracentrifugation to separate ribosomal subunits, monosomes, and polysomes

  • Western blot analysis of fractions to track RPS26 incorporation into various complexes

  • Quantitative analysis of free 40S/60S ratios as an indicator of assembly defects

• Immunoprecipitation studies:

  • Co-precipitation of RPS26 interacting partners during ribosome biogenesis

  • Time-course analysis to capture assembly intermediates

  • Comparison between wild-type and mutant RPS26 interaction networks

• Pulse-chase experiments:

  • Metabolic labeling combined with RPS26 immunoprecipitation

  • Analysis of RPS26 incorporation kinetics into nascent ribosomes

  • Assessment of how mutations affect incorporation timing or efficiency

• Fluorescence microscopy:

  • Track nucleolar localization of RPS26 during ribosome biogenesis

  • Co-localization with other assembly factors

  • Analysis of nucleolar-cytoplasmic shuttling dynamics

Research has shown that studies with Rps26a 5A mutant (five conserved residues within Y62–K70 segment replaced by alanines) revealed accumulation of free 60S subunits, suggesting defects in 40S formation or 80S assembly .

How can differences between human and yeast RPS26 be studied using antibodies?

Human and yeast RPS26 share significant homology but also exhibit important functional differences. Species-specific studies can provide insights into conserved and divergent aspects of RPS26 function:

• Cross-species complementation analysis:

  • Human RPS26 can partially complement yeast Δrps26a Δrps26b strains but with growth defects

  • Antibodies specific to human RPS26 can track the expressed human protein in yeast

• Structural and functional comparison:

  • Yeast strains expressing human RPS26 show altered 40S/60S ribosomal subunit ratios

  • Immunoprecipitation can identify differential binding partners between species

  • Western blot analysis can assess incorporation efficiency into yeast ribosomes

• Mutational analysis:

  • Creation of chimeric proteins combining domains from human and yeast RPS26

  • Antibodies recognizing species-specific epitopes can assess incorporation into ribosomes

  • Evaluation of which domains contribute most to functional differences

• Ribosome biogenesis kinetics:

  • Pulse-chase experiments comparing assembly rates between species

  • Evaluation of whether human RPS26 follows the same assembly pathway in yeast cells

  • Identification of rate-limiting steps in cross-species ribosome assembly

Why does RPS26 appear at 18-21 kDa in Western blots despite a calculated molecular weight of 13 kDa?

The discrepancy between RPS26's calculated molecular weight (13 kDa) and its observed migration pattern (18-21 kDa) is consistently reported across multiple studies . This anomalous migration can be attributed to several factors:

• Post-translational modifications: RPS26 may undergo modifications that increase its apparent molecular weight

• Protein characteristics: The highly basic nature of RPS26 affects SDS binding and electrophoretic mobility

• Structural features: Some regions of the protein may resist complete denaturation in SDS-PAGE

This pattern is reproducible and can actually serve as validation for antibody specificity. When troubleshooting, consider:

• Using 15% SDS-PAGE gels for better resolution of small proteins

• Including recombinant RPS26 as a size reference when available

• Confirming band identity through additional techniques such as mass spectrometry

• Noting that consistent observation of 18-21 kDa bands across multiple tissues and cell lines supports specific detection

What strategies can improve detection of RPS26 in immunohistochemistry applications?

Optimizing RPS26 detection in tissue sections requires attention to several critical parameters:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Extended retrieval time (20-30 minutes) may improve signal for difficult samples

• Antibody dilution and incubation:

  • Start with 1:50 dilution for paraffin sections

  • Overnight incubation at 4°C often provides better signal-to-noise ratio than shorter incubations

  • Consider signal amplification systems for low abundance detection

• Detection systems:

  • Polymer-based detection systems often provide better sensitivity than ABC methods

  • For fluorescence detection, try longer exposure times with lower antibody concentrations

• Tissue-specific considerations:

  • Human breast cancer and kidney tissues serve as reliable positive controls

  • Fixation time and processing should be optimized for each tissue type

  • Freshly cut sections (within 1-2 weeks) provide optimal staining results

• Background reduction:

  • Use 5-10% normal serum from secondary antibody species in blocking buffer

  • Include 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Consider commercial background-reducing reagents for challenging tissues