RPS6KA3 Antibody

What applications are most effective for detecting RPS6KA3 protein using antibodies?

RPS6KA3 (RSK2) antibodies have been validated for multiple research applications with varying sensitivity and specificity profiles. The most effective applications include Western Blotting (WB), Immunofluorescence (IF), Flow Cytometry (FCM), and ELISA .

For optimal detection specificity, the following application-specific dilution ranges are recommended:

The choice of application should be determined by your specific experimental objectives. Western blotting provides definitive molecular weight confirmation (80-83 kDa) , while immunofluorescence enables subcellular localization studies, particularly effective in models like HepG2 cells where distinct cytoplasmic and nuclear distributions have been documented .

How should RPS6KA3 antibodies be stored to maintain optimal activity?

Proper storage conditions are crucial for maintaining antibody functionality over time. RPS6KA3 antibodies require specific storage protocols depending on their formulation and frequency of use :

For long-term storage (>1 month):

• Store at -20°C in small working aliquots to prevent repeated freeze-thaw cycles

• Add cryoprotectants such as glycerol (final concentration 30-50%) for antibodies stored below -20°C

• Ensure airtight sealing to prevent evaporation and contamination

For short-term storage (≤1 month):

• Store at 4°C for frequent use applications

• Avoid exposure to direct light, particularly for conjugated antibodies

• Add sodium azide (0.03%) as a preservative for unconjugated antibodies, but note this may interfere with HRP-based detection systems

Experimental validation shows that properly stored RPS6KA3 antibodies maintain approximately 90% activity for 12 months when these conditions are followed .

What are the key considerations for validating RPS6KA3 antibody specificity?

Validation of RPS6KA3 antibody specificity is essential for robust experimental outcomes and should follow a systematic approach:

• Cross-reactivity assessment: While many commercial RPS6KA3 antibodies show specificity for human targets, cross-reactivity with other RSK family members (RSK1, RSK3, RSK4) should be experimentally verified through comparative analysis using recombinant proteins .

• Positive and negative control tissues/cells: HepG2 and HeLa cells demonstrate high endogenous RPS6KA3 expression and serve as effective positive controls for validation . Use tissues/cells with confirmed low expression or knockdown models as negative controls.

• Blocking peptide validation: Utilizing the immunogenic peptide fragment (such as AA 539-588) allows competitive binding assays to confirm specificity .

• Multiple detection methods: Confirm target detection using at least two independent techniques (e.g., western blot plus immunofluorescence).

• Band verification: The RPS6KA3 protein should appear as a distinct band at approximately 80-83 kDa in western blot applications .

How can phosphorylation-specific RPS6KA3 detection be optimized in signaling pathway studies?

Detecting specific phosphorylation states of RPS6KA3 requires careful experimental design due to the protein's complex activation patterns via multiple phosphorylation sites:

• Phospho-state preservation: Immediately process samples in phosphatase inhibitor cocktails containing sodium fluoride (10 mM), sodium orthovanadate (1 mM), and β-glycerophosphate (10 mM) to prevent dephosphorylation during extraction.

• Stimulation protocols: For mitogenic activation studies, optimal RPS6KA3 phosphorylation occurs with EGF treatment (100 ng/mL for 10 minutes) or PMA stimulation (100 nM for 30 minutes) in serum-starved cells .

• Phospho-specific antibody selection: Choose antibodies targeting key regulatory phosphorylation sites:

  • Thr577 (C-terminal kinase domain) for MAPK pathway activation

  • Ser386 (linker region) for PDK1-mediated activation

  • Ser227 (N-terminal kinase domain) for full enzymatic activation

• Dephosphorylation controls: Include lambda phosphatase-treated samples as negative controls to confirm phospho-specificity.

• Pathway inhibitor controls: Pre-treatment with MEK inhibitors (U0126, 10 μM) or PI3K inhibitors (LY294002, 20 μM) can provide pathway-specific controls for phosphorylation state analysis.

When implemented correctly, this approach allows quantitative assessment of RPS6KA3 activation dynamics in response to various stimuli and interaction with downstream substrates including NR4A1/NUR77 and ETV1/ER81 transcription factors .

What methodological approaches are most effective for studying RPS6KA3 subcellular localization?

RPS6KA3 exhibits dynamic subcellular localization patterns that reflect its activation state and function. Optimized methodological approaches include:

• Subcellular fractionation: Sequential extraction protocols using hypotonic lysis (cytoplasmic fraction), high-salt extraction (nuclear soluble fraction), and detergent treatment (nuclear insoluble/chromatin-bound fraction) allow biochemical quantification of RPS6KA3 distribution.

• Immunofluorescence microscopy: Fixed-cell immunostaining using RPS6KA3 antibodies (1:200-1:1000 dilution) alongside nuclear (DRAQ5) and cytoskeletal (fluorescent phalloidin) counterstains enables high-resolution visualization of localization patterns .

• Live-cell imaging: Expression of fluorescently-tagged RPS6KA3 (ensuring C-terminal tagging to preserve kinase function) allows real-time monitoring of translocation dynamics.

• Proximity ligation assays: For detecting RPS6KA3 interactions with spatially-defined partners, particularly within nuclear subdomains during transcriptional regulation.

Research has demonstrated that upon stimulation, RPS6KA3 rapidly translocates from cytoplasmic locations to the nucleus, where it phosphorylates and activates transcription factors including NR4A1/NUR77 . This translocation can be effectively visualized in HepG2 cells using immunofluorescence with RPS6KA3 antibodies .

What strategies can address cross-reactivity concerns between RPS6KA3 and other RSK family members?

The high sequence homology between RSK family members (RSK1-4) presents significant challenges for antibody specificity. Advanced strategies to ensure RPS6KA3-specific detection include:

• Epitope selection: Target unique regions of RPS6KA3, particularly in the linker domain (amino acids 350-400) or C-terminal region (amino acids 539-588), which show greater sequence divergence from other RSK isoforms .

• Immunodepletion approaches: Sequential immunoprecipitation with antibodies against potential cross-reactive proteins (RSK1, RSK3) prior to RPS6KA3 analysis.

• Knockout/knockdown validation: Utilize CRISPR/Cas9-generated RPS6KA3 knockout cells or siRNA-mediated knockdown to confirm antibody specificity through signal ablation.

• Isoform-specific blocking peptides: Competitive incubation with peptides corresponding to homologous regions of other RSK family members can identify cross-reactivity.

• Recombinant protein panels: Test antibody reactivity against purified recombinant RSK1-4 proteins to quantitatively assess cross-reactivity profiles.

When implementing these strategies, researchers should recognize that monoclonal antibodies typically offer higher specificity for RPS6KA3 detection compared to polyclonal alternatives, though at potentially reduced sensitivity .

How can RPS6KA3 antibodies be effectively utilized in protein-protein interaction studies?

RPS6KA3 functions through interactions with upstream activators, downstream substrates, and scaffold proteins. Optimizing antibody-based interaction studies requires:

• Co-immunoprecipitation optimization:

  • Use mild lysis conditions (1% NP-40 or 0.5% CHAPS) to preserve protein complexes

  • Pre-clear lysates with appropriate control IgG to reduce non-specific binding

  • Conjugate RPS6KA3 antibodies to solid supports (Protein A/G beads or magnetic beads) at 2-5 μg antibody per mg total protein

  • Include appropriate controls (IgG isotype, reciprocal IP)

• Proximity-based interaction detection:

  • BioID or TurboID approaches using RPS6KA3 fusion proteins to identify interaction neighborhoods

  • FRET/BRET assays for direct interaction monitoring in live cells

  • PLA (Proximity Ligation Assay) using RPS6KA3 antibodies paired with antibodies against putative interactors

• Endogenous interaction verification:

  • Stimulation conditions (e.g., EGF, insulin, PMA) can significantly alter interaction profiles

  • Phosphorylation-state specific antibodies can distinguish activation-dependent interactions

This methodological framework has successfully identified interactions between RPS6KA3 and transcription factors including CREB, ATF4, and histone deacetylases in response to mitogenic stimulation .

What are the most effective protocols for using RPS6KA3 antibodies in chromatin immunoprecipitation studies?

RPS6KA3 functions as a nuclear kinase regulating transcription factor activity and chromatin organization. Optimized chromatin immunoprecipitation (ChIP) protocols require:

• Crosslinking optimization:

  • Standard formaldehyde fixation (1% for 10 minutes) for protein-DNA interactions

  • Dual crosslinking (1 mM DSG followed by 1% formaldehyde) for improved capture of protein-protein-DNA complexes

• Sonication parameters:

  • Optimize to achieve 200-500 bp DNA fragments

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody selection and validation:

  • Use antibodies targeting the C-terminal domain of RPS6KA3 to avoid epitope masking during chromatin binding

  • Validate antibody efficacy in ChIP applications using known RPS6KA3-regulated promoters

• Technical considerations:

  • Include input controls (5-10% of starting material)

  • Use IgG negative controls matched to the host species of the RPS6KA3 antibody

  • Implement stringent washing conditions (high salt, LiCl) to reduce background

• Analysis approaches:

  • qPCR for targeted analysis of specific genomic regions

  • ChIP-seq for genome-wide binding profile analysis

This approach has been successfully employed to study RPS6KA3's role in regulating immediate early gene expression and chromatin modification at specific genomic loci in response to growth factor stimulation.

How can tissue-specific RPS6KA3 expression patterns be accurately characterized using immunohistochemistry?

Characterizing tissue-specific RPS6KA3 expression through immunohistochemistry requires careful optimization:

• Tissue preparation and antigen retrieval:

  • Formalin-fixed paraffin-embedded (FFPE) tissues require heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Fresh frozen tissues provide superior epitope preservation but require different fixation (4% paraformaldehyde)

• Antibody validation:

  • Validate staining patterns against tissues with known RPS6KA3 expression profiles

  • Include positive controls (brain tissue, particularly cerebellum and hippocampus)

  • Confirm specificity with blocking peptides or RPS6KA3-deficient tissues

• Detection systems:

  • Polymer-HRP systems offer superior sensitivity compared to biotin-streptavidin methods

  • Tyramide signal amplification provides enhanced detection for low-abundance expression

• Counterstaining considerations:

  • Nuclear counterstains (hematoxylin) aid in distinguishing nuclear vs. cytoplasmic RPS6KA3 localization

  • Double immunohistochemistry with cell-type specific markers enables identification of expressing cell populations

• Quantification approaches:

  • H-score methodology (intensity × percentage positive cells)

  • Digital image analysis with nuclei segmentation for quantitative assessment

These approaches have revealed differential RPS6KA3 expression across neural tissues, with particularly high expression in neurons of the hippocampus and cerebellum, consistent with its role in cognitive function and the pathophysiology of Coffin-Lowry syndrome .

What strategies can address weak or inconsistent RPS6KA3 detection in western blotting?

Weak or inconsistent RPS6KA3 signals in western blotting can be systematically addressed through the following optimization approaches:

• Sample preparation optimization:

  • Include phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) and protease inhibitors in lysis buffers

  • Use RIPA buffer with 0.1% SDS for improved extraction of nuclear-associated RPS6KA3

  • Heat samples at 70°C rather than 95°C to prevent aggregation of this large protein

• Electrophoretic separation:

  • Use lower percentage gels (8%) for better resolution of the 80-83 kDa RPS6KA3 protein

  • Extend transfer times (overnight at 30V) for efficient transfer of larger proteins

  • Consider wet transfer systems with 20% methanol for improved efficiency

• Detection optimization:

  • Extended primary antibody incubation (overnight at 4°C) at optimal dilution (1:500-1:2000)

  • Signal enhancement using high-sensitivity ECL substrates or signal amplification systems

  • PVDF membranes generally provide better retention of RPS6KA3 than nitrocellulose

• Positive controls:

  • Include lysates from cells with known high RPS6KA3 expression (HeLa, HepG2)

  • Consider using recombinant RPS6KA3 protein as a reference standard

• Verification strategies:

  • Use multiple antibodies targeting different epitopes of RPS6KA3

  • Confirm band identity through immunoprecipitation followed by western blotting

These approaches have been demonstrated to consistently detect the 80-83 kDa RPS6KA3 protein in various cell lines and tissue extracts .

How can non-specific background be minimized in immunofluorescence applications with RPS6KA3 antibodies?

Non-specific background is a common challenge in immunofluorescence applications with RPS6KA3 antibodies. Systematic optimization includes:

• Fixation and permeabilization optimization:

  • Compare paraformaldehyde (4%, 10 min) vs. methanol (-20°C, 10 min) fixation

  • Test different permeabilization reagents (0.1-0.5% Triton X-100, 0.1% saponin, or 0.05% Tween-20)

  • Optimize permeabilization time (5-15 minutes) based on cell type

• Blocking optimization:

  • Use species-appropriate serum (5-10%) matching the secondary antibody host

  • Add 1% BSA to reduce non-specific protein interactions

  • Include 0.1-0.3% Triton X-100 in blocking solutions for nuclear proteins

  • Extend blocking time to 1-2 hours at room temperature

• Antibody incubation:

  • Optimize primary antibody dilution (1:200-1:1000) and incubation time (overnight at 4°C)

  • Include 0.05% Tween-20 in antibody diluent to reduce non-specific binding

  • Implement stringent washing steps (3-5 washes, 5-10 minutes each)

• Control experiments:

  • Include secondary-only controls to assess non-specific secondary binding

  • Use peptide competition with the immunizing peptide to verify specificity

  • Compare staining patterns across multiple cell types with variable RPS6KA3 expression

• Signal-to-noise enhancement:

  • Use mounting media containing anti-fade reagents to preserve signal

  • Consider confocal microscopy for improved signal resolution and background reduction

These approaches have successfully visualized both cytoplasmic and nuclear localization of RPS6KA3 in various cell types, particularly in HepG2 cells following mitogenic stimulation .

How can flow cytometry protocols be optimized for intracellular RPS6KA3 detection?

Optimizing flow cytometry for intracellular RPS6KA3 detection requires specific technical considerations:

• Fixation and permeabilization:

  • Test multiple fixation approaches: 4% paraformaldehyde (10 min), 80% methanol (-20°C), or commercial fix/perm kits

  • For nuclear-localized RPS6KA3, use stronger permeabilization (0.1% Triton X-100 or saponin-based buffers)

  • Maintain consistent fixation times across experiments for reproducible results

• Antibody titration:

  • Perform formal titration experiments testing antibody concentrations from 1:100-1:800

  • Optimal signal-to-noise ratio typically occurs in the 1:200-1:400 range for RPS6KA3 antibodies

  • Include isotype controls at equivalent concentrations

• Signal amplification:

  • Consider biotin-streptavidin systems for low-abundance detection

  • Evaluate tandem dyes (PE-Cy7, APC-Cy7) for improved separation from autofluorescence

• Controls and validation:

  • Use stimulated vs. unstimulated cells to confirm detection of activation-dependent changes

  • Include fluorescence-minus-one (FMO) controls for accurate gating

  • Validate flow cytometry results against other methods (western blotting, immunofluorescence)

• Analytical considerations:

  • Gate on intact, single cells using FSC/SSC and singlet discrimination

  • Consider cell cycle analysis in parallel, as RPS6KA3 activation may vary across cell cycle phases

These approaches have successfully detected RPS6KA3 in various cell lines, with particularly robust signals observed in HepG2 cells following mitogenic stimulation .

How can multiplexed detection systems be utilized to study RPS6KA3 in complex signaling networks?

Understanding RPS6KA3's role within signaling networks requires simultaneous detection of multiple pathway components. Advanced multiplexing approaches include:

• Multi-color flow cytometry:

  • Combine RPS6KA3 detection with key pathway markers (phospho-ERK1/2, phospho-S6)

  • Use spectral compensation and fluorochrome selection to minimize spillover

  • Implement dimensionality reduction techniques (tSNE, UMAP) for population analysis

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies against RPS6KA3 and related pathway components

  • Enables simultaneous detection of 30+ parameters without fluorescence limitations

  • Particularly valuable for analyzing activation patterns across heterogeneous cell populations

• Multiplexed immunofluorescence:

  • Sequential immunostaining with tyramide signal amplification

  • Spectral imaging and unmixing for improved multiplexing capability

  • Consider cyclic immunofluorescence for ultra-high parameter imaging

• Multiplex western blotting:

  • Use size-separated multiplex detection with differently colored detection systems

  • Implement sequential reprobing protocols with complete stripping between antibodies

  • Consider automated western systems for improved reproducibility and quantification

These approaches enable correlation of RPS6KA3 activation state with upstream regulators and downstream effectors, providing a systems-level understanding of its signaling role in various biological contexts.

What considerations are important when developing RPS6KA3 proximity-based assays for high-throughput screening?

Proximity-based assays for RPS6KA3 provide powerful tools for high-throughput screening applications. Key development considerations include:

• FRET/BRET-based biosensor design:

  • Position fluorophores to minimize interference with kinase activity

  • Engineer sensor constructs that report on RPS6KA3 conformational changes upon activation

  • Include appropriate positive controls (constitutively active mutants) and negative controls (kinase-dead mutants)

• AlphaScreen/AlphaLISA development:

  • Optimize antibody pairs targeting phosphorylated substrates and total RPS6KA3

  • Determine optimal protein concentrations and hook points

  • Implement Z'-factor determination to assess assay robustness

• Cellular thermal shift assays (CETSA):

  • Optimize heating conditions specific to RPS6KA3 stability

  • Implement detection via western blotting or AlphaLISA formats

  • Validate with known RPS6KA3 inhibitors before screening campaigns

• Nanobret occupancy assays:

  • Generate NanoLuc-RPS6KA3 fusion constructs with preserved kinase function

  • Validate proper subcellular localization of fusion proteins

  • Optimize tracer molecule concentration and incubation conditions

These high-throughput approaches enable screening for RPS6KA3 modulators, facilitating the development of chemical probes and potential therapeutic agents targeting pathways dysregulated in diseases associated with aberrant RPS6KA3 activity.

How can genetic models be integrated with antibody-based detection for comprehensive RPS6KA3 functional studies?

Integrating genetic models with antibody-based detection creates powerful experimental systems for RPS6KA3 functional studies:

• CRISPR/Cas9-edited cell lines:

  • Generate RPS6KA3 knockout lines as negative controls for antibody validation

  • Create point mutants at key phosphorylation sites (T577A, S386A, S227A) to dissect regulation

  • Develop epitope-tagged knock-in lines (FLAG, HA) for improved detection specificity

• Inducible expression systems:

  • Tet-On/Off systems for controlled RPS6KA3 expression

  • Combine with antibodies against endogenous proteins to study dose-dependent effects

  • Implement rescue experiments in knockout backgrounds to establish causality

• Animal models:

  • RPS6KA3-deficient mouse models recapitulate aspects of Coffin-Lowry syndrome

  • Tissue-specific conditional knockout models paired with immunohistochemistry

  • Phospho-specific antibodies in genetic models to dissect in vivo signaling

• Patient-derived systems:

  • Antibody-based analysis in cells from Coffin-Lowry syndrome patients

  • iPSC models differentiated to relevant lineages for developmental studies

  • CRISPR correction of patient mutations to establish genotype-phenotype relationships

These integrated approaches provide powerful systems for dissecting RPS6KA3 function in physiological and pathological contexts, particularly in neurodevelopmental processes relevant to Coffin-Lowry syndrome .