RPS29 Antibody

What is RPS29 and why is it important in cellular function?

RPS29 (ribosomal protein S29) is a component of the 40S ribosomal subunit with a calculated molecular weight of 7 kDa. It consists of 56 amino acids and plays a crucial role in protein synthesis . Beyond its conventional role in ribosomes, RPS29 demonstrates important extra-ribosomal functions, including roles as an apoptosis inducer and RNase . Recent research has identified RPS29's involvement in protein degradation pathways, specifically targeting certain proteins for proteasomal degradation, which has significant implications for cellular regulatory mechanisms . RPS29 mutations have been linked to Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome, demonstrating its critical importance in hematopoiesis .

What are the recommended applications for RPS29 antibody?

RPS29 antibody has been validated for multiple experimental applications with specific recommended dilutions:

It is important to note that optimal dilutions are sample-dependent, and researchers should titrate the antibody in their specific testing systems to obtain optimal results . For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative approach .

What species reactivity can I expect with RPS29 antibody?

The RPS29 antibody (specifically referencing the 17374-1-AP variant) has been tested and confirmed to show reactivity with human and mouse samples . This cross-reactivity is consistent with the highly conserved nature of ribosomal proteins across species. When planning experiments with other species, preliminary validation is recommended as reactivity may vary between different antibody clones and manufacturers.

What is the molecular weight of RPS29 and how does this affect antibody detection?

RPS29 has a calculated molecular weight of 7 kDa (56 amino acids), and this observed molecular weight has been confirmed in experimental detection . When performing Western blot analysis, it's crucial to use appropriate percentage gels (typically 15-20% for such small proteins) and optimize transfer conditions for small proteins. The small size of RPS29 can present technical challenges in some applications, particularly in protein extraction and gel electrophoresis, where special care must be taken to prevent loss of small proteins during sample preparation.

How can I optimize RPS29 antibody for co-immunoprecipitation studies?

For effective co-immunoprecipitation (Co-IP) studies investigating RPS29 interactions, researchers should consider several optimization strategies. Based on published interactions between RPS29 and other proteins such as CYP6N3, a tandem affinity purification (TAP) approach can be particularly effective . In one validated protocol, researchers successfully used an N-terminally TAP-tagged RPS29 gene stably expressed from a pIB-V5-His expression vector .

For confirming protein interactions with RPS29, both in vitro and in vivo methods have been successful:

• GST pull-down assays for in vitro confirmation

• Immunofluorescence co-localization studies for in vivo validation

When conducting immunofluorescence to visualize RPS29 interactions, fluorescent tagging (e.g., GFP-tagged RPS29) combined with differently tagged interacting proteins (e.g., MYC-tagged interacting proteins) allows visualization of co-localization through confocal microscopy . In such experiments, RPS29 fluorescence has been observed distributed throughout the cell, with particular concentration in the cytoplasm where it may overlap with interacting partners .

What pre-rRNA processing defects are associated with RPS29 dysfunction, and how can they be assessed?

Research has established that mutations in RPS29 can cause pre-ribosomal RNA (rRNA) processing defects, particularly relevant in the context of Diamond-Blackfan anemia (DBA) . To assess these defects, researchers should consider:

• Northern blot analysis of pre-rRNA processing intermediates

• Quantitative RT-PCR to measure relative abundances of specific pre-rRNA species

• Next-generation sequencing approaches to comprehensively profile the rRNA processing landscape

When investigating DBA-associated mutations (such as p.I31F or p.I50T in RPS29), comparing pre-rRNA processing patterns between patient samples and healthy controls can reveal specific processing steps that are affected . The analysis should focus on both accumulation of precursor species and depletion of mature rRNAs, as both patterns provide insights into the stage at which processing is impaired.

In experimental models, haploinsufficiency of RPS29 (reduced expression to approximately 50% of normal levels) correlates with observable pre-rRNA processing defects, suggesting that quantitative rather than qualitative changes in RPS29 may drive pathology .

How does RPS29 antibody performance compare in different cell and tissue types?

The performance of RPS29 antibody varies across different cellular and tissue contexts, requiring specific optimization strategies. In Western blot applications, positive detection has been confirmed in human cell lines including HeLa and HepG2 cells . For immunohistochemistry, validation has been performed in mouse brain tissue .

When working with different tissue types, consider:

• Optimization of antigen retrieval methods: While TE buffer (pH 9.0) is recommended as the primary method for RPS29 antigen retrieval in IHC, citrate buffer (pH 6.0) provides an alternative that may perform better in certain tissues .

• Tissue-specific background: Ribosomal proteins are abundantly expressed across most cell types, which can lead to high background staining in certain tissues. Careful blocking and antibody dilution optimization are essential.

• Subcellular localization variation: While primarily observed in the cytoplasm, RPS29 distribution patterns may vary between cell types, especially considering its extra-ribosomal functions .

For neuronal tissues specifically, the recommended IHC dilution range starts at 1:50, potentially requiring adjustments toward the lower end of the dilution range (1:50-1:100) to achieve optimal signal-to-noise ratios .

What are the most effective protocols for using RPS29 antibody in Western blotting?

For optimal Western blot results with RPS29 antibody, researchers should follow these methodological considerations:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Consider phosphatase inhibitors if investigating potential phosphorylation states

  • For this small protein (7 kDa), avoid excessive heating during sample preparation

• Gel electrophoresis:

  • Use high percentage (15-20%) SDS-PAGE gels to properly resolve this small protein

  • Include appropriate molecular weight markers that cover the low molecular weight range

• Transfer conditions:

  • Optimize transfer time and voltage for small proteins (typically lower voltage for longer time)

  • Consider using PVDF membranes with 0.2 μm pore size rather than 0.45 μm for better retention of small proteins

• Antibody incubation:

  • Begin with a 1:1000-1:6000 dilution range, with 1:2000 as a recommended starting point

  • Incubate primary antibody at 4°C overnight for optimal results

  • Use 5% non-fat dry milk or BSA in TBST as blocking buffer

• Detection:

  • Enhanced chemiluminescence (ECL) systems are suitable for RPS29 detection

  • For quantitative analysis, consider fluorescent secondary antibodies

The observed molecular weight of RPS29 should be approximately 7 kDa, consistent with both calculated and experimentally verified weights .

How can I differentiate between specific and non-specific binding when using RPS29 antibody?

Distinguishing specific from non-specific binding is critical for reliable RPS29 antibody applications. Several methodological approaches can help ensure specificity:

• Positive and negative controls:

  • Use cell lines with confirmed RPS29 expression (HeLa, HepG2) as positive controls

  • Consider RPS29 knockdown or knockout samples as negative controls

  • For tissues, include isotype controls to assess non-specific binding

• Peptide competition assays:

  • Pre-incubate the antibody with excess RPS29 fusion protein or peptide immunogen

  • Compare staining patterns between competed and non-competed antibody

  • Specific signals should be significantly reduced or eliminated in competed samples

• Multiple antibody validation:

  • When possible, use multiple antibodies targeting different epitopes of RPS29

  • Consistent results across different antibodies increase confidence in specificity

• Molecular weight confirmation:

  • RPS29 should appear at 7 kDa on Western blots

  • Additional bands at significantly different molecular weights likely represent non-specific binding or degradation products

Recent advances in antibody specificity design using computational models may eventually provide even more specific RPS29 antibodies, as these approaches can disentangle multiple binding modes associated with specific ligands .

What are the most common technical challenges when using RPS29 antibody, and how can they be addressed?

Researchers frequently encounter several technical challenges when working with RPS29 antibody:

• Low signal intensity issues:

  • Challenge: RPS29's small size (7 kDa) can result in poor retention during transfer and processing

  • Solution: Use higher antibody concentration (starting at 1:1000 dilution), optimize transfer conditions for small proteins, and consider signal amplification methods

• High background in IHC applications:

  • Challenge: Ubiquitous ribosomal protein expression can lead to high background

  • Solution: Implement more stringent blocking (3-5% BSA), optimize antibody dilution (start at 1:50-1:100), and consider longer washing steps

• Inconsistent results between experiments:

  • Challenge: Variability in RPS29 detection between experimental replicates

  • Solution: Standardize all protocol steps, including sample preparation, antibody incubation times/temperatures, and detection methods

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with other ribosomal proteins

  • Solution: Validate antibody specificity using knockout/knockdown controls and peptide competition assays

• Epitope masking in complex samples:

  • Challenge: RPS29 interactions with other proteins may mask antibody epitopes

  • Solution: Optimize extraction and denaturation conditions, consider alternative epitope retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

For IHC specifically, antigen retrieval optimization is critical, with suggested protocols using either TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0 as alternatives .

How can RPS29 antibody be used to study Diamond-Blackfan anemia (DBA) mechanisms?

RPS29 antibody serves as a valuable tool for investigating Diamond-Blackfan anemia (DBA) mechanisms, following the identification of RPS29 mutations as a novel cause of autosomal dominant DBA . When designing experiments to study DBA using RPS29 antibody, researchers should consider:

• Expression analysis in patient samples:

  • Western blot analysis using RPS29 antibody can confirm haploinsufficiency in DBA patients with RPS29 mutations

  • Compare protein levels between patients with known RPS29 mutations (p.I31F or p.I50T) and healthy controls

  • Quantitative analysis should reveal approximately 50% reduction in RPS29 protein levels in affected individuals

• Pre-rRNA processing assessment:

  • RPS29 antibody can be used in RNA immunoprecipitation studies to identify RPS29-associated pre-rRNA species

  • Compare pre-rRNA processing patterns between normal and mutant RPS29-expressing cells

  • Correlate processing defects with erythroid differentiation abnormalities

• Zebrafish model investigations:

  • Using rps29−/− zebrafish models, RPS29 antibody can help validate phenotypes and rescue experiments

  • Immunohistochemistry applications (1:50-1:500 dilution) can assess tissue-specific expression patterns during development

• Erythroid differentiation studies:

  • Implement RPS29 antibody in flow cytometry or immunofluorescence to track expression during erythroid differentiation stages

  • Correlate RPS29 levels with markers of effective erythropoiesis in normal versus DBA models

This methodological approach provides a comprehensive framework for elucidating RPS29's role in DBA pathophysiology, focusing on both the primary ribosomal defects and downstream consequences for erythroid development.

What is known about the role of RPS29 in protein degradation pathways, and how can antibody-based methods help investigate this?

Research has revealed that RPS29 plays a significant role in protein degradation pathways, particularly through the proteasome system. Studies have demonstrated that RPS29 can target specific proteins for proteasomal degradation, representing an important extra-ribosomal function . To investigate this role using antibody-based approaches, researchers should consider:

• Co-immunoprecipitation studies:

  • Use RPS29 antibody to pull down protein complexes

  • Analyze associated proteins, particularly those involved in ubiquitin-proteasome system

  • For example, research has identified interaction between RPS29 and CYP6N3, with RPS29 promoting CYP6N3 degradation via the proteasome

• Proteasome inhibition experiments:

  • Treat cells with proteasome inhibitors (e.g., MG132, bortezomib)

  • Use RPS29 antibody in Western blot to assess changes in RPS29 levels and its interaction partners

  • This approach can help distinguish between proteasome-dependent and independent effects of RPS29

• Immunofluorescence co-localization studies:

  • Combine RPS29 antibody with antibodies against proteasome components or ubiquitinated proteins

  • Assess co-localization patterns, particularly under conditions of cellular stress

  • Previous studies have used this approach to visualize RPS29 distribution throughout the cell and its overlap with interacting partners in the cytoplasm

• Quantitative analysis of target protein degradation:

  • Use Western blot with RPS29 antibody alongside antibodies for suspected target proteins

  • Compare protein levels in RPS29-overexpressing, normal, and RPS29-depleted conditions

  • For instance, RPS29 overexpression has been shown to increase CYP6N3 protein degradation, while RPS29 knockdown stabilizes CYP6N3

This multi-faceted approach provides a comprehensive framework for investigating RPS29's role in protein degradation pathways across different experimental contexts.

How can computational modeling enhance RPS29 antibody specificity for research applications?

Recent advances in computational modeling offer promising approaches to enhance RPS29 antibody specificity. Biophysics-informed models can identify distinct binding modes associated with specific ligands, enabling the design of antibodies with customized specificity profiles . For researchers looking to implement these approaches for RPS29 antibody optimization:

• Binding mode identification strategies:

  • Apply computational models to disentangle multiple binding modes

  • Train biophysics-informed models on experimentally selected antibodies

  • Associate each potential ligand with a distinct binding mode to enable prediction and generation of specific variants

• High-throughput sequencing integration:

  • Combine phage display experiments with high-throughput sequencing

  • Perform selections against various combinations of ligands to build training and test sets

  • Use these datasets to build and assess computational models for antibody specificity

• Specificity profile customization:

  • Design antibodies with either specific high affinity for RPS29 or cross-specificity for RPS29 and related proteins

  • Optimize energy functions associated with each binding mode to generate novel antibody sequences with predefined binding profiles

  • For cross-specific sequences, jointly minimize functions associated with desired ligands; for specific sequences, minimize functions for desired ligands while maximizing those for undesired ligands

These computational approaches provide powerful tools for designing antibodies with desired specificity profiles beyond what can be achieved through selection alone, potentially yielding RPS29 antibodies with unprecedented specificity and cross-reactivity characteristics.

What novel research applications are emerging for RPS29 antibody beyond traditional protein detection?

Beyond traditional protein detection applications, RPS29 antibody is finding novel applications in cutting-edge research areas:

• Therapeutic target validation:

  • RPS29's role in Diamond-Blackfan anemia makes it a potential therapeutic target

  • RPS29 antibody can help validate small molecule or gene therapy approaches targeting RPS29 expression or function

  • Assessment of drug effects on RPS29 levels and associated pathways in pre-clinical models

• Stress response and ribosome biogenesis studies:

  • RPS29 antibody can track changes in ribosome composition during cellular stress

  • Investigating how RPS29 levels, localization, and interactions change under different stress conditions

  • Correlation of these changes with alterations in translation efficiency and specificity

• Extracellular vesicle (EV) cargo analysis:

  • Recent interest in ribosomal proteins as components of EV cargo

  • RPS29 antibody can help characterize EV content and potential signaling roles

  • Investigation of RPS29-containing EVs as biomarkers in various pathological conditions

• Post-translational modification mapping:

  • Combining RPS29 antibody with mass spectrometry to identify and map post-translational modifications

  • Understanding how these modifications affect RPS29 function in both ribosomal and extra-ribosomal contexts

  • Correlation of modification patterns with cellular states and disease conditions

• Structural biology applications:

  • Using RPS29 antibody fragments as crystallization chaperones

  • Facilitating structural determination of RPS29-containing complexes

  • Providing insights into the structural basis of RPS29's diverse functional roles

These emerging applications highlight the expanding utility of RPS29 antibody beyond conventional detection methods, opening new avenues for understanding fundamental biological processes and disease mechanisms.