RPS6KA3 Antibody, HRP conjugated

What is RPS6KA3 and what are its major biological functions?

RPS6KA3 (Ribosomal Protein S6 Kinase, 90kDa, Polypeptide 3) is a serine/threonine-protein kinase that functions downstream of ERK (MAPK1/ERK2 and MAPK3/ERK1) signaling. The protein mediates mitogenic and stress-induced activation of transcription factors including CREB1, ETV1/ER81, and NR4A1/NUR77. RPS6KA3 plays critical roles in regulating translation through RPS6 and EIF4B phosphorylation and mediates cellular proliferation, survival, and differentiation by modulating mTOR signaling. Additionally, it represses the pro-apoptotic function of BAD and DAPK1, promoting cell survival . In fibroblasts, RPS6KA3 is specifically required for EGF-stimulated phosphorylation of CREB1 and histone H3 at 'Ser-10', leading to transcriptional activation of immediate-early genes .

What applications is the HRP-conjugated RPS6KA3 antibody specifically optimized for?

The HRP-conjugated RPS6KA3 antibody is primarily optimized for ELISA applications as indicated in multiple product listings . The horseradish peroxidase conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps. This makes the antibody particularly valuable for high-throughput screening applications and quantitative protein detection assays. While the unconjugated versions of RPS6KA3 antibodies support multiple applications including Western Blotting, Immunohistochemistry, and Immunofluorescence , the HRP-conjugated version is specifically engineered to maximize sensitivity in ELISA-based detection systems.

What is the optimal storage condition for maintaining RPS6KA3 antibody activity?

For optimal maintenance of activity, RPS6KA3 antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt . To minimize activity loss from repeated freeze-thaw cycles, it is recommended to aliquot the antibody before freezing . For working solutions, short-term storage at 4°C is suitable, but extended storage should be at recommended freezing temperatures. The antibody is typically preserved in a buffer containing 0.03% Proclin 300 and 50% Glycerol in 0.01M PBS, pH 7.4, which helps maintain stability during storage .

How should positive and negative controls be selected for experiments using RPS6KA3 antibody?

For positive controls, select tissues or cell lines known to express RPS6KA3, such as human fibroblasts which utilize this kinase in EGF signaling pathways . When designing experiments with phospho-specific RPS6KA3 antibodies (such as pTyr529), include samples treated with growth factors that activate the MAPK pathway (e.g., EGF, PMA) as positive controls . For negative controls, consider using:

• Primary antibody omission controls

• Isotype controls (rabbit IgG with the same concentration)

• Tissues or cell lines with known RPS6KA3 knockdown/knockout

• Preabsorption with immunizing peptide when available

Additionally, for phospho-specific antibodies, include samples treated with relevant kinase inhibitors or phosphatase treatments as technical negative controls .

What are the recommended protocols for using RPS6KA3 antibody, HRP conjugated in ELISA applications?

For optimal results in ELISA applications using HRP-conjugated RPS6KA3 antibody, follow this methodological approach:

• Coating: Coat ELISA plates with target antigen or capture antibody (2-10 μg/ml in carbonate buffer pH 9.6) and incubate overnight at 4°C

• Blocking: Block with 2-5% BSA or non-fat milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature

• Sample addition: Add samples containing RPS6KA3 protein and incubate for 1-2 hours at room temperature

• Antibody incubation: Dilute HRP-conjugated RPS6KA3 antibody (CSB-PA020466LB01HU) to optimal working concentration (typically 1:1000 to 1:5000) in blocking buffer and incubate for 1 hour at room temperature

• Detection: Add TMB substrate and incubate for 15-30 minutes protected from light, then stop reaction with 2N H₂SO₄

• Readout: Measure absorbance at 450nm with reference at 620nm

For sandwich ELISA, it's recommended to pair the HRP-conjugated antibody with a compatible unconjugated antibody targeting a different epitope of RPS6KA3 .

What optimization steps are needed when transitioning from unconjugated to HRP-conjugated RPS6KA3 antibody?

When transitioning from unconjugated to HRP-conjugated RPS6KA3 antibody, researchers should implement the following optimization steps:

• Dilution series testing: Perform a titration experiment with dilutions ranging from 1:100 to 1:10,000 to determine optimal antibody concentration that maximizes signal-to-background ratio

• Incubation time adjustment: HRP-conjugated antibodies typically require shorter incubation times (30-60 minutes) compared to two-step detection systems

• Buffer optimization: Test the antibody in different diluents (PBS-T with varying percentages of BSA or non-fat milk) to minimize background

• Substrate selection: Optimize substrate choice based on detection sensitivity requirements (TMB for colorimetric, luminol-based for chemiluminescence)

• Blocking agent compatibility: Ensure blocking reagent doesn't cross-react with HRP enzyme

Additionally, researchers should validate that the HRP conjugation hasn't affected epitope recognition by comparing detection patterns with unconjugated versions across different sample preparations .

How can researchers address non-specific binding issues with RPS6KA3 antibody, HRP conjugated?

To address non-specific binding issues with RPS6KA3 antibody, HRP conjugated, implement the following methodological approaches:

• Optimize blocking conditions: Test different blocking agents (BSA, non-fat milk, casein, or commercial blocking buffers) at concentrations between 2-5%

• Increase washing stringency: Add additional washing steps and increase Tween-20 concentration in washing buffer to 0.1-0.3%

• Antibody diluent optimization: Include 0.1-0.5% Tween-20 and/or 0.1-1% BSA in antibody diluent to reduce non-specific interactions

• Cross-adsorption: If cross-reactivity with related proteins is suspected, pre-incubate antibody with the purified cross-reactive protein

• Sample pre-clearing: Pre-clear samples by incubation with isotype control antibodies bound to beads

• HRP inhibitor screening: Test samples for endogenous peroxidase activity and include appropriate quenching steps if necessary

These approaches should be implemented sequentially, analyzing results after each modification to identify the most effective strategy for reducing non-specific binding while maintaining specific signal detection .

What criteria should be used to interpret RPS6KA3 phosphorylation data in signaling pathway analysis?

When interpreting RPS6KA3 phosphorylation data in signaling pathway analysis, researchers should apply the following critical evaluation criteria:

• Temporal dynamics assessment: Analyze phosphorylation kinetics at multiple time points (5, 15, 30, 60 minutes) following stimulus application to capture both rapid and sustained responses

• Multi-site phosphorylation evaluation: Compare phosphorylation patterns across key sites including Tyr529, Thr573, and Ser227, as these sites have different functional implications

• Pathway inhibitor controls: Include data from samples treated with specific inhibitors (MEK, ERK, mTOR inhibitors) to confirm pathway specificity

• Functional correlation: Correlate phosphorylation levels with downstream functional outcomes (e.g., CREB1 activation, BAD inhibition)

• Quantitative analysis: Apply densitometry analysis with appropriate normalization to total RPS6KA3 protein levels

A comprehensive interpretation should consider that pTyr529 phosphorylation generally indicates activation within the ERK signaling cascade, while the functional consequences may depend on cell type and concurrent signaling events .

What are the most common technical artifacts encountered with HRP-conjugated antibodies and how can they be distinguished from true signals?

Common technical artifacts encountered with HRP-conjugated antibodies and strategies to distinguish them from true signals include:

These artifacts can be systematically evaluated through carefully designed control experiments that isolate each variable, enabling researchers to distinguish technical issues from biological phenomena .

How can RPS6KA3 phosphorylation status be effectively monitored in response to therapeutic interventions?

To effectively monitor RPS6KA3 phosphorylation in response to therapeutic interventions, researchers should implement a multi-faceted approach:

• Multiplex phosphorylation profiling: Use combinatorial antibody approaches to simultaneously detect multiple phosphorylation sites (pTyr529, pThr573, pSer227) to generate a comprehensive activation profile

• Temporal resolution optimization: Establish time-course experiments with appropriate sampling intervals based on the pharmacokinetics of the therapeutic agent

• Single-cell analysis integration: Complement bulk analyses with phospho-flow cytometry or immunofluorescence using specific phospho-antibodies to assess cell-to-cell variability in response

• Pathway context evaluation: Assess RPS6KA3 phosphorylation in context with upstream (ERK1/2) and downstream (CREB, BAD) effectors to understand signaling dynamics

• Quantitative Western blot analysis: Implement fluorescence-based quantitative Western blotting with appropriate loading controls and standard curves

For therapeutic agents targeting the MAPK pathway, monitoring RPS6KA3 phosphorylation provides critical insights into pathway modulation and potential resistance mechanisms. Combining phospho-specific antibodies with total protein detection allows calculation of the phosphorylation ratio, which serves as a more reliable biomarker than absolute phosphorylation levels .

What are the considerations for using RPS6KA3 antibodies in studies of Coffin-Lowry syndrome?

When using RPS6KA3 antibodies in studies of Coffin-Lowry syndrome (CLS), researchers should consider the following critical factors:

• Mutation-specific antibody selection: Choose antibodies that recognize epitopes outside common mutation regions in CLS patients, as documented in the RPS6KA3 gene (mutations are associated with CLS)

• Domain-specific detection: Select antibodies targeting different domains (N-terminal vs. C-terminal) to distinguish potentially truncated protein products

• Cell type considerations: Focus on neuronal cell types and tissues relevant to CLS pathophysiology, as RPS6KA3 function in neuronal development is particularly relevant to this syndrome

• Patient sample variability: Account for the heterogeneity of mutations across CLS patients by designing experiments that can detect varying levels of protein expression or truncated forms

• Functional correlation approaches: Combine protein detection with functional assays measuring kinase activity to correlate molecular findings with disease severity

For CLS research specifically, it's valuable to use both phospho-specific and total protein antibodies to determine if mutant RPS6KA3 proteins retain partial functionality or display altered phosphorylation patterns that might contribute to disease mechanisms .

What emerging techniques are enhancing the utility of RPS6KA3 antibodies in spatial proteomics?

Several emerging techniques are significantly enhancing the utility of RPS6KA3 antibodies in spatial proteomics research:

• Proximity ligation assays (PLA): This technique enables visualization of protein-protein interactions involving RPS6KA3 at the single-molecule level. Recent applications have employed RPS6KA3 antibodies in PLA to map interaction networks in the MAPK signaling cascade, revealing context-dependent binding partners across subcellular compartments

• Multiplexed immunofluorescence: Advanced multiplexing approaches using cyclic immunofluorescence or spectral unmixing allow simultaneous detection of RPS6KA3 alongside multiple signaling components. This approach has revealed previously unrecognized spatial organization of signaling complexes in neuronal cells

• Super-resolution microscopy integration: Combining RPS6KA3 antibodies with techniques like STORM or PALM has enabled nanoscale mapping of kinase distribution, particularly in dendritic spines where RPS6KA3 plays critical roles in synaptic plasticity

• Mass spectrometry immunohistochemistry (MSIHC): This hybrid approach combines antibody-based protein localization with mass spectrometry identification, allowing precise mapping of RPS6KA3 and its post-translational modifications across tissue sections with subcellular resolution

• Spatial transcriptomics correlation: Integrating RPS6KA3 protein detection with spatial transcriptomics provides multi-omic insights into regional activation patterns and regulatory mechanisms controlling protein expression and activity

These methodological advances are particularly valuable for understanding the compartmentalized signaling of RPS6KA3 in complex tissues like brain, where spatial organization significantly impacts signaling outcomes .

How do different commercial sources of RPS6KA3 antibodies compare in specificity and sensitivity?

Comparative analysis of RPS6KA3 antibodies from different commercial sources reveals important differences in specificity and sensitivity:

For optimal experimental outcomes, researchers should select antibodies based on their specific application requirements, considering factors such as phosphorylation status detection, species reactivity, and detection method compatibility. Independent validation using genetic knockdown/knockout controls is recommended to confirm specificity regardless of commercial source .

What validation standards should be applied when using RPS6KA3 antibodies in novel cell types or tissues?

When using RPS6KA3 antibodies in novel cell types or tissues, researchers should apply the following comprehensive validation standards:

• Expression verification: Confirm RPS6KA3 expression in the target tissue/cell type through complementary methods such as RT-PCR, RNA-seq, or proteomics data

• Molecular weight confirmation: Verify that detected bands match the expected molecular weight (90 kDa for full-length RPS6KA3) through Western blot analysis

• Knockout/knockdown controls: When possible, include negative controls using CRISPR/Cas9 knockout or siRNA knockdown samples to confirm signal specificity

• Signal induction: Demonstrate appropriate signal modulation after relevant stimuli (e.g., growth factors that activate the MAPK pathway) or inhibitors

• Cross-reactivity assessment: Perform peptide competition assays to confirm epitope specificity, particularly important when working with closely related RSK family members

• Subcellular localization pattern: Verify that the observed subcellular distribution matches known localization patterns of RPS6KA3

• Phospho-antibody responsiveness: For phospho-specific antibodies, demonstrate appropriate signal changes following phosphatase treatment

These validation steps should be systematically documented and included in research publications to establish antibody reliability in the specific experimental context .

How can discrepancies between RPS6KA3 protein levels and functional activity be reconciled in experimental data?

Discrepancies between RPS6KA3 protein levels and functional activity can be reconciled through a multi-parameter analysis approach:

• Comprehensive phosphorylation profiling: Assess multiple phosphorylation sites (including pTyr529, pThr573, pSer227) simultaneously, as RPS6KA3 requires phosphorylation at multiple residues for full activation

• Inhibitor protein interaction analysis: Evaluate interactions with regulatory proteins that might inhibit kinase activity without affecting protein levels

• Subcellular fractionation studies: Analyze RPS6KA3 distribution across subcellular compartments, as localization significantly impacts functional activity

• Direct kinase activity assays: Complement protein detection with in vitro kinase assays using immunoprecipitated RPS6KA3 and appropriate substrates

• Proteomic profiling of post-translational modifications: Identify additional modifications (ubiquitination, SUMOylation) that might impact protein activity

• Protein conformation analysis: Consider techniques that can detect conformational changes indicative of activation state

This integrated approach recognizes that protein abundance alone is insufficient to predict functional activity, particularly for kinases like RPS6KA3 that undergo complex regulatory processes. By simultaneously assessing protein levels, phosphorylation status, and enzymatic activity, researchers can develop more accurate models of RPS6KA3 function in their experimental systems .

What emerging applications of RPS6KA3 antibodies are relevant to precision medicine approaches?

Emerging applications of RPS6KA3 antibodies in precision medicine include:

• Biomarker development: RPS6KA3 phosphorylation status is being investigated as a potential biomarker for MAPK pathway activation in cancer, with particular relevance to predicting response to MEK/ERK inhibitors. Phospho-specific antibodies are crucial for developing immunohistochemistry-based companion diagnostics

• Neurological disorder stratification: Given RPS6KA3's established role in Coffin-Lowry syndrome and emerging connections to other neurological conditions, antibody-based detection methods are being developed to characterize patient-specific molecular phenotypes for potential therapeutic stratification

• Therapeutic resistance monitoring: RPS6KA3 activation represents a potential resistance mechanism to targeted therapies. Monitoring its phosphorylation status during treatment could identify adaptive signaling pathway activation, enabling timely intervention with combination therapies

• Scaffold-mediated signaling analysis: Newer applications focus on using proximity ligation assays with RPS6KA3 antibodies to map signaling complexes, which could reveal patient-specific differences in signaling network architecture relevant to drug response

• Single-cell profiling for heterogeneity assessment: Integration of RPS6KA3 antibodies in single-cell proteomic approaches is enabling characterization of intrinsic resistance populations within tumors, potentially allowing more precise therapeutic targeting

These applications represent the evolving interface between basic RPS6KA3 biology and clinical applications, where antibody-based detection technologies serve as critical translational tools .

How might advances in antibody engineering improve RPS6KA3 detection and functional analysis?

Recent and anticipated advances in antibody engineering that will improve RPS6KA3 detection and functional analysis include:

• Conformation-specific antibodies: Development of antibodies that specifically recognize active versus inactive conformations of RPS6KA3, enabling direct visualization of kinase activation states without relying solely on phosphorylation markers

• Intrabodies and nanobodies: Engineered smaller antibody formats capable of functioning within living cells to track RPS6KA3 dynamics in real-time, potentially revealing previously unobservable activation patterns and protein interactions

• Split-fluorescent protein complementation tags: Antibody fragments fused to split fluorescent proteins for direct visualization of RPS6KA3 interactions with specific binding partners in living systems

• Bispecific antibodies for signaling cascade analysis: Dual-targeting antibodies designed to simultaneously recognize RPS6KA3 and its upstream activators or downstream substrates, providing direct evidence of functional pathway connectivity

• Internalization-capable antibody conjugates: Development of antibody formats that can be internalized by living cells to detect intracellular RPS6KA3, allowing longitudinal studies of signaling dynamics

• Modular recombinant antibody systems: Platforms enabling rapid epitope switching while maintaining detection modality, facilitating comprehensive mapping of RPS6KA3 functional domains

These technological advances will significantly expand the research toolkit beyond current antibody capabilities, transforming RPS6KA3 research from primarily static observations to dynamic functional analysis .

What novel insights might be gained from integrating RPS6KA3 antibody-based detection with other 'omics technologies?

Integration of RPS6KA3 antibody-based detection with complementary 'omics technologies offers several promising avenues for novel scientific insights:

• Phosphoproteomics integration: Correlating antibody-detected RPS6KA3 activation with global phosphoproteomic profiles can reveal previously unrecognized downstream substrates and signaling branches. This approach has already identified novel connections between RPS6KA3 and chromatin remodeling complexes in neuronal differentiation models

• Spatial transcriptomics correlation: Combining RPS6KA3 protein localization data with spatial transcriptomics is revealing how localized kinase activity shapes regional gene expression patterns, particularly in complex tissues like brain where RPS6KA3 mutations cause Coffin-Lowry syndrome

• Single-cell multi-omics: Integration of antibody-based detection at the single-cell level with transcriptomics and metabolomics is uncovering cell state-dependent activation patterns and unexpected heterogeneity in RPS6KA3 signaling outcomes

• Interactome mapping with proteomics: Utilizing RPS6KA3 antibodies for immunoprecipitation followed by mass spectrometry is revealing context-dependent interaction networks that explain tissue-specific functions of this widely expressed kinase

• Chromatin immunoprecipitation with sequencing (ChIP-seq): Applying RPS6KA3 antibodies in ChIP-seq experiments is identifying direct roles in chromatin regulation beyond its classical cytoplasmic signaling functions