RPS29 Antibody, HRP conjugated

What is RPS29 and what is its cellular function?

RPS29 (Ribosomal Protein S29) functions as a component of the small ribosomal subunit (40S). The protein is essential for the ribonucleoprotein complex responsible for cellular protein synthesis. Multiple studies have confirmed its role in translation, with PubMed literature demonstrating its presence in the small ribosomal subunit . The ribosomal protein contributes to the structural integrity of the 40S subunit and participates in protein translation mechanisms. RPS29 is also referred to in scientific literature as "Small ribosomal subunit protein uS14" or "40S ribosomal protein S29," reflecting its localization and function within the ribosomal machinery .

Recent research has expanded our understanding beyond RPS29's canonical role in translation, revealing potential regulatory functions in metabolic pathways and protein-protein interactions that influence cellular processes beyond protein synthesis. This multifunctional nature makes RPS29 antibodies valuable tools for investigating both ribosomal and extra-ribosomal functions.

What are the key specifications of commercially available RPS29 Antibody, HRP conjugated?

The commercially available RPS29 antibody with HRP conjugation typically demonstrates the following specifications:

• Binding Specificity: Amino acids 2-56 of the RPS29 protein

• Reactivity: Primarily human samples, with some antibodies showing cross-reactivity with mouse, rat, and other species

• Host: Rabbit (for polyclonal variants)

• Purification: >95% purity, Protein G purified

• Immunogen: Recombinant Human 40S ribosomal protein S29 protein (2-56AA)

• Isotype: IgG

• Applications: ELISA primarily, with some variants suitable for Western Blot (WB) and Immunofluorescence (IF)

These specifications ensure researchers can select appropriate antibodies based on their specific experimental requirements for detecting and studying RPS29 in various applications.

How should researchers validate the specificity of RPS29 Antibody, HRP conjugated?

Validation of RPS29 antibody specificity should follow a multi-step approach to ensure reliable experimental results:

• Western Blot Analysis: Run a western blot using protein extracts from cells known to express RPS29 (such as human cell lines). The antibody should detect a single band at approximately 6.7 kDa, which corresponds to the molecular weight of RPS29.

• Positive and Negative Controls: Include known positive controls (human samples) and negative controls (samples from species with low homology or RPS29-knockdown cells) to confirm specificity.

• Immunoprecipitation Validation: Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is capturing RPS29 specifically.

• Immunofluorescence Pattern Analysis: The staining pattern should be consistent with ribosomal localization, showing primarily cytoplasmic distribution with potential nucleolar presence during ribosome biogenesis .

• Blocking Peptide Competition: Pre-incubate the antibody with the immunizing peptide (amino acids 2-56) to demonstrate that this eliminates specific binding in your assay.

These validation steps ensure that experimental results obtained with the RPS29 antibody are both reliable and reproducible, which is crucial for research integrity.

What techniques can effectively study RPS29 protein interactions?

Several sophisticated techniques have been successfully employed to study RPS29 protein interactions:

• Tandem Affinity Purification (TAP): This method has been particularly effective for identifying RPS29 binding partners under native conditions. The technique employs a dual tag system (such as streptavidin and calmodulin binding peptide) separated by a TEV protease cleavage site, allowing for sequential purification steps that yield high specificity .

• GST Pull-Down Assays: This technique provides confirmation of direct protein-protein interactions without cofactors. Studies have successfully used GST-tagged RPS29 immobilized on glutathione sepharose resin to capture interaction partners such as CYP6N3 .

• Immunofluorescence and Confocal Microscopy: Co-localization studies using tagged proteins (such as GFP-tagged RPS29 and MYC-tagged proteins of interest) can visualize interactions in cellular contexts. Research has shown that RPS29 fluorescence is distributed throughout the cell while interacting with cytoplasmic proteins .

• Fluorescence Resonance Energy Transfer (FRET): This technique allows for detection of protein interactions in living cells with high sensitivity. Studies have successfully employed RFP-tagged proteins and GFP-tagged RPS29 to demonstrate interactions in vivo .

• Co-immunoprecipitation: This approach can confirm interactions by precipitating RPS29 along with its binding partners from cell lysates.

Each technique offers distinct advantages for investigating different aspects of RPS29 interactions, with complementary approaches providing the most robust evidence of biological relevance.

How does RPS29 regulate metabolic insecticide resistance through CYP6N3 interaction?

Research has revealed a novel regulatory mechanism whereby RPS29 modulates insecticide resistance through direct interaction with CYP6N3, a cytochrome P450 family member. The mechanism involves several key steps:

• Direct Binding: RPS29 directly binds to CYP6N3 as demonstrated by GST pull-down assays, immunofluorescence, and FRET analysis .

• Post-translational Regulation: RPS29 overexpression results in a dose-dependent decrease in CYP6N3 protein levels without affecting CYP6N3 mRNA expression, indicating post-translational regulation .

• Proteasomal Degradation Pathway: Treatment with MG132 (a proteasome inhibitor) prevents RPS29-mediated reduction in CYP6N3 levels, confirming that RPS29 targets CYP6N3 for proteasomal degradation .

• Functional Consequence: CYP6N3 overexpression enhances resistance to deltamethrin (DM) in mosquito cells, while RPS29 overexpression counteracts this effect by reducing CYP6N3 protein levels .

• Reciprocal Regulation: Interestingly, CYP6N3 overexpression increases RPS29 expression levels, suggesting a complex feedback mechanism .

This regulatory pathway represents a novel mechanism for modulating insecticide resistance and potentially offers new targets for pest control strategies.

What are the optimal conditions for using RPS29 Antibody, HRP conjugated in ELISA assays?

For optimal ELISA performance with RPS29 Antibody, HRP conjugated, researchers should consider the following protocol parameters:

Coating Buffer: Carbonate-bicarbonate buffer (pH 9.6) typically yields optimal antigen binding.

Antigen Concentration: For recombinant RPS29 protein, a concentration range of 1-5 μg/ml is recommended for coating.

Blocking Solution: 3-5% BSA in PBS provides effective blocking while maintaining low background.

Antibody Dilution: The HRP-conjugated RPS29 antibody typically performs optimally at dilutions between 1:500 and 1:2000, though specific optimization is recommended for each lot .

Incubation Conditions:

• Primary incubation: 2 hours at room temperature or overnight at 4°C

• Wash steps: 4-5 washes with PBS-T (0.05% Tween-20)

• Substrate incubation: 15-30 minutes at room temperature in the dark

Detection Substrate: TMB (3,3',5,5'-Tetramethylbenzidine) substrate is recommended for HRP detection, with reaction stopping using 2N H₂SO₄.

Controls: Include both positive controls (known RPS29-containing samples) and negative controls (buffer only and non-specific protein) to validate assay specificity.

Careful optimization of these parameters will ensure maximum sensitivity and specificity for RPS29 detection in ELISA formats.

How can researchers overcome common challenges when using RPS29 Antibody in immunohistochemistry?

Researchers frequently encounter challenges when using RPS29 antibodies for immunohistochemistry. Here are evidence-based solutions:

• High Background Signal:

  • Increase blocking time to 2 hours using 5% normal serum from the same species as the secondary antibody

  • Optimize antibody dilution (1:1000 dilution has been successful for paraffin-embedded human pancreas tissue)

  • Use 0.3% H₂O₂ in methanol for peroxidase blocking prior to primary antibody incubation

• Weak or Absent Signal:

  • Implement heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Extend primary antibody incubation to overnight at 4°C

  • Use signal amplification systems such as avidin-biotin complex (ABC) or tyramide signal amplification

• Non-specific Staining:

  • Pre-adsorb the antibody with tissue powder

  • Include 0.1-0.3% Triton X-100 in the antibody diluent for better penetration

  • Validate specificity using RPS29 knockdown controls

• Inconsistent Results Between Experiments:

  • Standardize fixation times and conditions

  • Prepare fresh working solutions for each experiment

  • Include positive control tissues in each staining run

Successful staining of paraffin-embedded human pancreas tissue has been achieved using ab254638 at 1/1000 dilution, providing a useful reference point for optimization .

What are the advantages and limitations of using HRP-conjugated versus unconjugated RPS29 antibodies?

Understanding the comparative advantages and limitations of HRP-conjugated versus unconjugated RPS29 antibodies is crucial for experimental design:

Advantages of HRP-Conjugated RPS29 Antibodies:

• Streamlined Workflow: Eliminates the need for secondary antibody incubation, reducing protocol time by approximately 1-2 hours .

• Reduced Background: Minimizes non-specific binding associated with secondary antibodies, particularly beneficial in tissues with endogenous immunoglobulins.

• Consistent Signal: Provides a defined 1:1 ratio of enzyme to antibody, improving quantitative reliability.

• Multiplexing Capability: Facilitates dual labeling with antibodies from the same host species.

Limitations of HRP-Conjugated RPS29 Antibodies:

• Reduced Sensitivity: Generally less sensitive than detection systems utilizing unconjugated primary antibodies with amplification steps.

• Limited Signal Amplification: Cannot benefit from signal enhancement provided by multiple secondary antibodies binding to a single primary antibody.

• Shorter Shelf Life: Conjugated antibodies typically have reduced stability compared to unconjugated variants.

• Application Restrictions: Primarily optimized for ELISA applications, while unconjugated antibodies demonstrate broader utility across ELISA, WB, IHC, IF, and ICC applications .

Comparative Application Suitability:

Selection should be based on specific experimental requirements, with HRP-conjugated antibodies preferred for rapid ELISA protocols and unconjugated variants for applications requiring maximum sensitivity or versatility.

How can researchers effectively use RPS29 antibodies to investigate ribosome biogenesis defects?

Investigating ribosome biogenesis defects using RPS29 antibodies requires specialized approaches:

• Nucleolar Stress Monitoring:

  • Use immunofluorescence with RPS29 antibodies to track nucleolar-cytoplasmic shuttling during stress conditions

  • Compare normal localization patterns with redistributions occurring during biogenesis defects

  • Implement co-staining with nucleolar markers (fibrillarin, nucleolin) to assess nucleolar integrity

• Pre-ribosomal Complex Analysis:

  • Perform sucrose gradient fractionation followed by western blotting with RPS29 antibodies

  • Monitor shifts in RPS29 distribution between free protein, pre-40S particles, and mature ribosomes

  • Compare fractionation profiles between normal and biogenesis-defective conditions

• Pulse-Chase Experiments:

  • Use metabolic labeling with 35S-methionine combined with immunoprecipitation using RPS29 antibodies

  • Track incorporation of RPS29 into nascent ribosomes over time

  • Identify delays or defects in pre-ribosome maturation

• Protein-Protein Interaction Networks:

  • Implement proximity ligation assays (PLA) with RPS29 antibodies and antibodies against other ribosomal processing factors

  • Quantify interaction changes during normal biogenesis versus defective conditions

  • Map interaction networks affected by specific biogenesis defects

• Co-localization Studies:

  • Perform high-resolution imaging using RPS29 antibodies alongside markers for different phases of ribosome biogenesis

  • Quantify co-localization coefficients to detect subtle changes in the assembly process

  • Track trajectories of RPS29-containing particles during export from the nucleus

These methodological approaches provide complementary data for comprehensive analysis of ribosome biogenesis defects, with RPS29 serving as an informative marker for small subunit assembly and maturation.

What is the significance of RPS29's role in protein degradation pathways beyond ribosomal function?

Recent research has revealed that RPS29 possesses significant extraribosomal functions, particularly in protein degradation pathways. This represents an emerging area of investigation with several important implications:

• Novel Regulatory Mechanism: RPS29 has been demonstrated to target specific proteins, such as CYP6N3, for proteasomal degradation. This occurs through direct protein-protein interaction rather than through ribosome-mediated mechanisms . This finding challenges the traditional view of ribosomal proteins as solely structural components of the translation machinery.

• Post-translational Control: Studies have shown that RPS29 overexpression results in a dose-dependent decrease in CYP6N3 protein levels without affecting CYP6N3 mRNA expression . This post-translational regulation provides an additional layer of control for protein expression beyond transcriptional and translational regulation.

• Pathway Specificity: The degradation appears to be mediated through the ubiquitin-proteasome pathway, as treatment with MG132 (a proteasome inhibitor) prevents RPS29-mediated reduction in protein levels . This suggests RPS29 may function within specific degradation pathways rather than as a general mediator of protein turnover.

• Physiological Relevance: In the case of CYP6N3, this degradation mechanism modulates insecticide resistance, demonstrating a practical significance of this extraribosomal function . This raises questions about what other physiological processes might be regulated by similar RPS29-mediated mechanisms.

• Evolution of Ribosomal Protein Functions: This represents an example of functional diversification of ribosomal proteins throughout evolution, potentially revealing how ancient cellular components have acquired new roles.

These findings open new research directions for investigating how ribosomal proteins might function in coordinating translation with protein degradation, potentially serving as integrated regulators of proteostasis.

How do researchers effectively differentiate between canonical and non-canonical functions of RPS29 in experimental settings?

Differentiating between RPS29's canonical ribosomal functions and its non-canonical roles requires sophisticated experimental approaches:

• Ribosome Profiling Combined with RPS29 Manipulation:

  • Compare ribosome occupancy and translation efficiency using ribosome profiling in cells with wild-type versus mutated RPS29

  • Identify transcripts differentially affected by RPS29 mutations that disrupt either ribosomal or extraribosomal functions

• Domain-Specific Mutations:

  • Generate RPS29 variants with mutations in regions critical for ribosome incorporation versus regions involved in protein-protein interactions

  • Test these variants for rescue of different phenotypes to separate translation-dependent from translation-independent functions

• Polysome Association Analysis:

  • Perform subcellular fractionation to separate polysome-associated RPS29 from free RPS29

  • Compare interaction partners between these fractions using co-immunoprecipitation followed by mass spectrometry

• Temporal Protein Expression Control:

  • Implement rapid induction systems for RPS29 expression and monitor immediate effects (likely extraribosomal) versus delayed effects (potentially ribosome-mediated)

  • Use pulse-chase experiments to track the timing of phenotypic changes relative to ribosome assembly kinetics

• Targeted Degradation Approaches:

  • Employ techniques like Trim-Away to rapidly degrade endogenous RPS29

  • Compare acute effects (likely representing non-canonical functions) with longer-term effects (representing disruption of translation)

These methodological approaches provide complementary strategies for dissecting the multiple functions of RPS29, allowing researchers to attribute specific cellular effects to either canonical or non-canonical activities.

What future research directions might emerge from current understandings of RPS29 function and interactions?

Based on current findings, several promising research directions are emerging for RPS29 investigation:

• Therapeutic Targeting of RPS29-Mediated Protein Degradation:

  • Development of small molecules that modulate RPS29's ability to target specific proteins for degradation

  • Exploration of this pathway as a novel approach for degrading disease-relevant proteins resistant to conventional therapeutics

• Systems Biology Analysis of RPS29 Interaction Networks:

  • Comprehensive mapping of the RPS29 interactome across different cell types and conditions

  • Network analysis to identify cellular pathways coordinately regulated by RPS29 through both ribosomal and extraribosomal mechanisms

• Role in Stress Response and Cellular Adaptation:

  • Investigation of how RPS29-mediated protein degradation might be regulated during cellular stress responses

  • Examination of potential roles in coordinating translation inhibition with selective protein degradation during adaptation to changing environments

• Evolutionary Conservation of Extraribosomal Functions:

  • Comparative analysis of RPS29 functions across species to determine when the protein degradation role emerged

  • Identification of structural adaptations that enabled acquisition of extraribosomal functions

• Potential Roles in Human Disease:

  • Investigation of RPS29 dysregulation in disorders characterized by protein homeostasis defects

  • Exploration of genetic variations in RPS29 that might affect its extraribosomal functions without disrupting translation

• Agricultural Applications:

  • Development of novel insecticide resistance management strategies based on modulation of the RPS29-CYP6N3 regulatory axis

  • Screening for compounds that enhance RPS29-mediated degradation of insecticide resistance factors

These research directions represent exciting opportunities for expanding our understanding of RPS29 biology beyond its canonical role in translation, potentially leading to novel therapeutic and agricultural applications.