Phospho-RPS6 (Ser235) Antibody

What is Phospho-RPS6 (Ser235) Antibody and why is it important in cell signaling research?

Phospho-RPS6 (Ser235) Antibody is a research tool designed to specifically detect the phosphorylated form of the S6 Ribosomal Protein at serine 235. This antibody is crucial for studying cellular signaling pathways as ribosomal protein S6 (RPS6) is a major component of the small 40S ribosomal subunit implicated in mRNA decoding . RPS6 serves as a critical downstream substrate of multiple signaling cascades, including mTORC1 and MAPK/ERK pathways.

Methodologically, this antibody enables researchers to:

• Monitor activation of the mTOR and ERK signaling pathways

• Assess translational regulation in response to various stimuli

• Examine cell growth and proliferation mechanisms

• Evaluate metabolic responses in different experimental conditions

The significance lies in that RPS6 phosphorylation enhances its affinity for the m7GpppG cap complex, promoting translation initiation and increasing cellular protein synthesis capacity .

How does RPS6 phosphorylation occur and what sites are important?

Phosphorylation of RPS6 follows a hierarchical and sequential pattern:

• Initial phosphorylation begins at Ser-236

• Sequential phosphorylation then occurs at Ser-235, Ser-240, Ser-244, and Ser-247

This ordered phosphorylation process is regulated by at least two distinct kinase families:

The dual phosphorylation at Ser235/236 can occur through both mTORC1-dependent mechanisms via p70 S6 kinase (S6K1) and mTORC1-independent mechanisms via p90 ribosomal S6 kinases (RSK) activated by extracellular signal-regulated kinases (ERK) . This dual regulation makes these sites particularly informative for monitoring multiple signaling inputs.

What are the optimal experimental applications for Phospho-RPS6 (Ser235) antibodies?

Based on validated research applications, Phospho-RPS6 (Ser235) antibodies can be utilized in multiple experimental techniques:

For optimal results in western blotting, it is recommended to use treatment conditions that activate mTOR or ERK pathways, such as insulin stimulation, IGF-1 treatment, or Calyculin A (phosphatase inhibitor) treatment . When performing immunofluorescence or flow cytometry, treatment with 100 nM Calyculin A for 30 minutes has been validated to produce optimal signal .

How can I validate the specificity of Phospho-RPS6 (Ser235) antibodies?

Proper validation of phospho-specific antibodies is critical. Follow these methodological approaches:

• Phosphatase treatment controls:

  • Split your sample and treat half with lambda phosphatase

  • The phospho-signal should be eliminated in the treated sample

• Stimulation/inhibition experiments:

  • Stimulate cells with insulin, IGF-1, or serum to increase phosphorylation

  • Treat cells with mTOR inhibitors (rapamycin, Torin1) or MEK inhibitors (U0126, PD0325901) to decrease phosphorylation

  • Compare treated vs. untreated samples by western blotting

• Mutation analysis:

  • Use cells expressing Ser235/236Ala mutant RPS6 as negative controls

  • This approach has been validated for testing antibody specificity

• Peptide competition:

  • Pre-incubate antibody with immunizing phospho-peptide

  • Signal should be blocked in subsequent applications

Research has demonstrated that introducing individual Ser → Ala mutations at codons 235, 236, 240, 244, and 247 of RPS6 and expressing the mutant proteins in cells provides excellent specificity controls for phospho-specific antibodies .

How does the sequential phosphorylation of RPS6 affect its function in translation regulation?

The hierarchical phosphorylation of RPS6 creates a sophisticated regulatory mechanism:

• Cap-binding enhancement: Phosphorylation enhances RPS6 affinity for the m7GpppG cap, facilitating translation initiation complex assembly

• Bidirectional influence: Research indicates a bidirectional interaction where:

  • Phospho-Ser-240 and phospho-Ser-244 promote phosphorylation of Ser-247

  • Phospho-Ser-247 in turn promotes phosphorylation of Ser-240 and Ser-244

• Selective mRNA translation: Phosphorylated RPS6 preferentially enhances translation of:

  • mRNAs containing 5' terminal oligopyrimidine (TOP) tracts

  • mRNAs encoding ribosomal proteins and translation factors

• Cellular outcomes: The level of RPS6 phosphorylation directly impacts:

  • Cell size regulation

  • Global protein synthesis capacity

  • Glucose homeostasis in pancreatic β-cells

Notably, mutation of Ser-247 inhibits phosphorylation of Ser-240 and Ser-244 but has no effect on phosphorylation of Ser-235/236, suggesting independent regulatory mechanisms for different phosphorylation clusters .

What are the distinctions between mTORC1-dependent and ERK-dependent phosphorylation of RPS6?

Understanding the dual regulation of RPS6 phosphorylation is essential for experimental interpretation:

Methodological approach to distinguish between pathways:

• Use rapamycin (mTORC1 inhibitor) to block S6K-mediated phosphorylation

• Use MEK inhibitors to block RSK-mediated phosphorylation

• Examine temporal dynamics - ERK/RSK activation is typically faster than mTORC1/S6K

• Monitor additional pathway-specific substrates (e.g., 4E-BP1 for mTORC1)

The phosphorylation of RPS6 at Ser235/236 can occur independently of mTORC1 via RSK, making these sites less specific markers for mTORC1 activity compared to Ser240/244 phosphorylation .

How can Phospho-RPS6 antibodies be utilized in cancer research and therapeutic development?

Phospho-RPS6 antibodies serve as valuable tools in cancer research:

• Biomarker applications:

  • Hyperphosphorylation of RPS6 has been documented in multiple cancer types

  • Monitor treatment efficacy of mTOR inhibitors in clinical specimens

  • Predict sensitivity to targeted therapies in patient-derived samples

• Resistance mechanism studies:

  • Identify bypass mechanisms in mTOR inhibitor-resistant tumors

  • Map cross-talk between PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways

  • Detect feedback activation of alternative signaling routes

• Methodology for therapeutic evaluation:

  • Use standardized immunohistochemistry (IHC) protocols (1:300-1:1200 dilution)

  • Implement multi-parameter flow cytometry to measure pathway activation in heterogeneous tumor populations

  • Combine with other pathway markers for comprehensive signaling analysis

When analyzing clinical samples, researchers should standardize tissue handling procedures, as phospho-epitopes can be rapidly lost during sample processing. Immediate fixation and validated extraction protocols are essential for reliable results.

What are the most effective protocols for using Phospho-RPS6 antibodies in flow cytometry experiments?

Optimized protocol for flow cytometry with Phospho-RPS6 antibodies:

• Cell preparation:

  • Culture cells under desired conditions (with/without stimulation)

  • For optimal signal, treat cells with 100 nM Calyculin A for 30 minutes prior to fixation

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with True-Phos™ Perm Buffer or 90% ice-cold methanol for 30 minutes

• Antibody staining:

  • Block with 2% BSA in PBS for 30 minutes

  • Use ≤0.25 μg Phospho-RPS6 antibody per million cells in 100 μl volume

  • Incubate for 45-60 minutes at room temperature

• Detection and analysis:

  • For direct detection, use fluorochrome-conjugated antibodies (PE, BV421, PE/Cy7)

  • For indirect detection, use appropriate secondary antibodies

  • Include isotype controls and unstimulated/inhibitor-treated cells as controls

For multi-parameter analysis, compatible fluorochrome-conjugated Phospho-RPS6 antibodies include PE, PE/Cyanine7, PerCP/Cyanine5.5, and Brilliant Violet 421™, allowing flexibility in panel design .

How do I address common challenges when using Phospho-RPS6 antibodies in experimental settings?

For western blotting applications, researchers should note that the observed molecular weight of RPS6 is approximately 32-35 kDa, which is slightly higher than the calculated molecular weight of 29 kDa due to post-translational modifications .

When troubleshooting immunofluorescence experiments, remember that different fixation methods can affect epitope accessibility. Paraformaldehyde fixation (4%, 10-15 minutes) followed by permeabilization with Triton X-100 (0.1%, 5 minutes) generally provides good results for phospho-epitopes .

What are the critical considerations for storing and handling Phospho-RPS6 antibodies?

Proper storage and handling are essential for maintaining antibody performance:

• Storage conditions:

  • Store antibodies at -20°C to -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

  • Some formulations contain 50% glycerol allowing storage at -20°C without freezing solid

• Buffer composition:

  • Most antibodies are provided in PBS with stabilizers such as:

    • 50% glycerol

    • 0.02-0.09% sodium azide

    • 0.1-0.5% BSA

• Stability considerations:

  • Working dilutions should be prepared fresh

  • Most antibodies remain stable for at least one year after shipment when properly stored

  • Some products may not require aliquoting for -20°C storage

• Handling precautions:

  • Avoid contamination by using sterile technique

  • Protect conjugated antibodies from prolonged light exposure

  • Centrifuge vials briefly before opening to collect solution at the bottom

Following manufacturer recommendations for specific products is always advisable, as formulations may vary between suppliers.

What are emerging applications of Phospho-RPS6 antibodies in single-cell analysis and spatial biology?

Recent advancements have expanded the utility of Phospho-RPS6 antibodies:

• Single-cell phospho-profiling:

  • Mass cytometry (CyTOF) incorporates metal-tagged Phospho-RPS6 antibodies

  • Single-cell RNA-seq combined with protein epitope profiling (CITE-seq) allows correlation of phosphorylation state with transcriptome

  • High-dimensional flow cytometry panels incorporate up to 30 parameters including multiple phospho-proteins

• Spatial phospho-proteomics:

  • Multiplexed immunofluorescence with tyramide signal amplification enhances sensitivity

  • Imaging mass cytometry allows subcellular localization of phosphorylated RPS6

  • Digital spatial profiling correlates phospho-signal with tissue microenvironment

• Live-cell dynamics:

  • Proximity ligation assays monitor real-time changes in RPS6 phosphorylation

  • Phospho-specific intrabodies track pathway activation in living cells

These emerging methodologies allow researchers to move beyond bulk analysis to understand signaling heterogeneity at single-cell resolution and within spatial contexts, providing deeper insights into pathway regulation in complex tissues and tumors.

How can Phospho-RPS6 antibodies be integrated into multi-omics research approaches?

Integration of Phospho-RPS6 analysis into multi-omics workflows:

• Proteogenomic integration:

  • Correlate RPS6 phosphorylation with:

    • Mutational status of pathway components (mTOR, PI3K, PTEN, etc.)

    • Expression of upstream regulators

    • Translation efficiency of specific mRNA subsets

• Metabolomics connections:

  • Link RPS6 phosphorylation status to:

    • Amino acid availability and utilization

    • Energy metabolites (ATP/AMP ratio)

    • Lipid metabolism signatures

• Methodological approach:

  • Fixed/frozen sample splitting for parallel omics analysis

  • Single-cell multi-omics to correlate phospho-status with other molecular features

  • Sequential antibody stripping and reprobing for multiplexed phospho-profiling

• Data integration frameworks:

  • Pathway analysis incorporating phosphorylation events

  • Correlation networks linking phospho-signals to transcriptome and metabolome

  • Machine learning approaches to identify predictive phospho-signatures

The integration of phospho-specific antibody data with other omics approaches enables systems-level understanding of how RPS6 phosphorylation impacts cellular homeostasis across various physiological and pathological conditions.

How do different Phospho-RPS6 antibodies compare in specificity and performance?

When selecting between these options:

• For mTORC1 pathway specificity, choose Ser240/244 antibodies

• For detecting both mTORC1 and MAPK/ERK inputs, use Ser235/236 antibodies

• For distinguishing between single vs. dual phosphorylation events, compare Ser235-specific with Ser235/236 antibodies

• For quantitative applications like flow cytometry, monoclonal antibodies generally provide more consistent results

Research has demonstrated that monoclonal antibodies offer higher reproducibility across experiments, while polyclonal antibodies may provide higher sensitivity but with greater lot-to-lot variation .

What are the advantages of recombinant versus conventional antibody production for Phospho-RPS6 detection?

Comparing recombinant and conventional antibody production methods:

Several commercially available Phospho-RPS6 antibodies now utilize recombinant technology, including:

• Recombinant rabbit monoclonal antibodies for improved reproducibility

• Single-chain variable fragments (scFvs) for enhanced tissue penetration

• Engineered antibody formats for specialized applications

Methodologically, researchers should consider:

• Recombinant antibodies for longitudinal studies requiring consistent reagents

• Recombinant technology for rare phospho-epitopes difficult to raise conventionally

• Engineered formats for challenging applications (intracellular delivery, multiplexing)

The transition to recombinant antibody technology represents an important advancement in phospho-specific detection, addressing many limitations of traditional antibody production methods.

What is the current consensus on best practices for Phospho-RPS6 antibody usage in translational research?

Based on extensive research experience and published literature, these best practices are recommended:

• Selection criteria:

  • Choose antibodies validated in your specific application

  • Select phosphorylation sites appropriate for your research question (pathway-specificity)

  • Consider using multiple antibodies targeting different phosphorylation sites for comprehensive analysis

• Experimental design:

  • Always include appropriate positive controls (stimulated samples)

  • Incorporate negative controls (phosphatase-treated, inhibitor-treated, non-phosphorylatable mutants)

  • Standardize sample collection and processing to minimize phospho-epitope loss

• Reporting standards:

  • Document complete antibody information (manufacturer, catalog number, lot, dilution)

  • Report RRID (Research Resource Identifier) for antibodies (e.g., AB_2834769, AB_2918654)

  • Describe detailed methodology including fixation, permeabilization, and detection systems

• Validation requirements:

  • Demonstrate specificity through multiple approaches (western blot, phosphatase treatment)

  • Confirm expected molecular weight (~32 kDa for RPS6)

  • Validate response to known pathway activators and inhibitors

These consensus guidelines ensure robust, reproducible results when using Phospho-RPS6 antibodies in translational research settings.

What future developments can we anticipate in Phospho-RPS6 antibody technology and applications?

Emerging trends and future directions include:

• Advanced antibody engineering:

  • Bispecific antibodies detecting multiple phosphorylation states simultaneously

  • Antibody-based biosensors for real-time pathway monitoring

  • Enhanced membrane-permeable formats for live-cell applications

• Expanded clinical applications:

  • Standardized IHC protocols for patient stratification

  • Companion diagnostics for mTOR/PI3K pathway inhibitors

  • Minimally invasive monitoring of treatment response

• Technology integration:

  • AI-assisted image analysis for quantitative phospho-profiling

  • Automated high-content screening platforms

  • Point-of-care phospho-protein testing for personalized medicine

• Novel detection modalities:

  • CRISPR-based detection systems linked to phospho-recognition domains

  • Digital protein quantification with single-molecule resolution

  • Label-free detection systems for dynamic pathway analysis