RPS6 Antibody

Basic Research Questions

• What is RPS6 and why is it important in cell signaling research?

  Ribosomal Protein S6 (RPS6) is a crucial component of the 40S ribosomal subunit involved in protein synthesis. It serves as a major substrate for S6 kinases, which phosphorylate it in response to various extracellular signals . This phosphorylation is essential for controlling protein translation and consequently cellular function and metabolism. RPS6 plays a vital role in regulating cell growth and proliferation, making it an important marker in signaling pathway research .

  RPS6 is highly conserved across eukaryotic species, underscoring its fundamental role in ribosomal function and cellular biology. It is commonly used as a readout of mammalian target of rapamycin complex 1 (mTORC1) activity, making it valuable for studying cellular responses to growth factors, nutrients, and stress conditions .

• How do I select the appropriate RPS6 antibody for my specific application?

  When selecting an RPS6 antibody, consider these key factors:

  • Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, etc.)

  • Species reactivity: Ensure the antibody recognizes RPS6 in your target species

  • Clone specificity: For monoclonal antibodies, check which epitope they recognize (e.g., C-8 targets the N-terminal epitope comprising amino acids 1-249 of human RPS6)

  • Technical considerations: For IHC applications with mouse samples, consider that mouse monoclonal antibodies may cause mouse-on-mouse (MOM) background staining issues

  Example of application-specific performance:

  Application	Recommended Dilution	Validated Antibodies
  Western Blot	1:500-1:1000	MAB5436, ab127980, sc-74459
  IHC-P	1:50-1:200	5G10 Rabbit mAb (#2217)
  ICC/IF	1:100-1:400	ab40820, ab127980
  IP	Validated	sc-74459, ab70227

• What controls should I use when working with RPS6 antibodies?

  Proper controls are essential for RPS6 antibody experiments:

  • Positive control: Use cell lines known to express RPS6, such as HeLa, HepG2, or NIH-3T3

  • Negative control: RPS6 knockout or knockdown cells can serve as specificity controls

  • Loading control: For Western blots, use established housekeeping proteins

  • Treatment controls: For phospho-specific antibodies, include samples treated with pathway inhibitors (e.g., rapamycin to inhibit mTOR pathway)

  Search result mentions validation using Western blot with both wildtype and RPS6 knockout cells to confirm antibody specificity: "Total lysates (15 μg protein) from 293T and 293T/RPS6 CRISPR knockout cell lines were used to validate specificity."

Advanced Research Questions

• How can I differentiate between different phosphorylation states of RPS6 in my experimental system?

  RPS6 contains multiple phosphorylation sites that can be selectively detected:

  • Use phospho-specific antibodies that recognize distinct sites (S235/236, S240/244)

  • Employ pharmacological inhibitors to manipulate specific kinase pathways

  • Consider using phosphatase treatments as controls

  • For comprehensive analysis, combine immunoblotting with mass spectrometry

  Research has shown that phosphorylation at S235, S236, S240, and S244 upregulates the translation of mRNAs containing an oligopyrimidine tract at their transcriptional start sites . This phosphorylation is stimulated by growth factors, tumor promoting agents, and other mitogens.

  In a study examining trastuzumab resistance, researchers found that p-rpS6 activity remained unchanged in resistant cells following treatment, while it was suppressed in parental cell lines . The study demonstrated this through both immunoblotting and immunocytochemistry techniques.

• What considerations should I make when using RPS6 antibodies in mouse models of ribosomal protein deficiency?

  When studying RPS6 in mouse models with altered ribosomal protein expression:

  • Consider using heterozygous models, as complete RPS6 knockout is lethal

  • Be aware that RPS6 hemizygous mice develop specific hematopoietic phenotypes including macrocytic anemia and lymphopenia

  • Monitor elevated eADA (erythrocyte adenosine deaminase) levels (2.30±0.11 vs. 1.13±0.02 in controls)

  • Use antibodies validated specifically for mouse tissues

  Research with Rps6 hemizygous mice has shown they develop hypoproliferative, macrocytic anemia, granulocytopenia, thrombocytosis, and lymphopenia . This makes them valuable models for studying how ribosomal protein insufficiency affects cellular function, particularly in conditions like Diamond-Blackfan anemia.

• How can RPS6 antibodies be used to monitor mTOR pathway activity in therapeutic contexts?

  RPS6 phosphorylation serves as a reliable downstream indicator of mTOR pathway activity:

  • Use phospho-specific RPS6 antibodies to assess response to mTOR inhibitors

  • Monitor therapy efficacy in cancer treatment protocols

  • Track changes in pathway activation during drug resistance development

  • Correlate p-RPS6 levels with clinical outcomes

  Research indicates that p-rpS6 could serve as a potential molecular marker to predict individual patient responsiveness to therapies targeting signaling pathways . In trastuzumab-resistant breast cancer cells, p-rpS6 levels inversely correlated with drug-induced growth inhibition when treated with HER2-targeting drugs, suggesting its utility as a post-treatment indicator of resistance.

Methodological Questions

• What is the optimal protocol for detecting phosphorylated RPS6 in clinical samples?

  For reliable detection of phosphorylated RPS6 in clinical samples:

  1. Sample collection: Rapidly preserve specimens to prevent dephosphorylation

  2. Fixation: Use phosphatase inhibitors during sample preparation

  3. Antibody selection: Choose phospho-specific antibodies with clinical validation

  4. Detection method: Consider using amplification systems for enhanced sensitivity

  5. Quantification: Use digital imaging and analysis software for objective assessment

  In a study examining trastuzumab resistance biomarkers, researchers performed immunocytochemistry by incubating cells with p-rpS6 (S235/236) antibody and observed higher fluorescent intensity among resistant sublines compared to parental lines . The technique confirmed that treatment with trastuzumab did not decrease p-rpS6 expression in resistant cells but did so in parental cells.

• How should I optimize Western blot conditions for RPS6 antibody detection?

  For optimal Western blot detection of RPS6:

  1. Sample preparation: Use RIPA buffer with proteinase and phosphatase inhibitors

  2. Protein loading: Load 10-20 μg/lane of whole cell lysates

  3. Gel selection: Use gradient Tris-glycine gels for optimal separation

  4. Transfer conditions: Transfer to nitrocellulose membrane

  5. Blocking: Use 5% non-fat milk or BSA in TBST

  6. Antibody dilution: Typically 1:500-1:1000 for primary antibodies

  7. Detection method: Use appropriate secondary antibodies and detection systems

  Example protocol from search result : "PVDF membrane was probed with 0.25 μg/mL of Mouse Anti-Human/Mouse/Rat Ribosomal Protein S6 Monoclonal Antibody (Catalog # MAB5436) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Ribosomal Protein S6 at approximately 32 kDa."

• What are the recommended protocols for immunofluorescence using RPS6 antibodies?

  For immunofluorescence detection of RPS6:

  1. Fixation: Use 4% paraformaldehyde or methanol fixation

  2. Permeabilization: 0.1-0.5% Triton X-100 in PBS

  3. Blocking: 5-10% normal serum or BSA

  4. Primary antibody: Dilute to 1:100-1:400 and incubate overnight at 4°C or 2-3 hours at room temperature

  5. Secondary antibody: Use fluorophore-conjugated secondaries appropriate for your microscopy setup

  6. Nuclear counterstain: DAPI is commonly used

  Example protocol from search result : "Ribosomal Protein S6/RPS6 was detected in immersion fixed MM55K mouse kidney cell line using Mouse Anti-Human/Mouse/Rat Ribosomal Protein S6/RPS6 Monoclonal Antibody (Catalog # MAB5436) at 25 μg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm."

Research Application Questions

• How are RPS6 antibodies used in cancer research to study drug resistance mechanisms?

  RPS6 antibodies provide valuable insights into cancer drug resistance mechanisms:

  • Monitor changes in mTOR pathway activation during treatment

  • Identify shifts in translational regulation in resistant cells

  • Evaluate correlation between RPS6 phosphorylation status and therapeutic response

  • Assess post-translational modifications as potential biomarkers

  A study on trastuzumab resistance in breast cancer demonstrated that phosphorylated RPS6 levels remained unchanged in resistant cells following treatment, while they decreased in sensitive parental cell lines . The researchers found that p-rpS6 levels inversely correlated with drug-induced growth inhibition when resistant cells were treated with HER2-targeting drugs, suggesting p-rpS6 could serve as a potential molecular marker for predicting therapeutic responsiveness.

• What is the significance of RPS6 in ribosome biogenesis disorders and related disease models?

  RPS6 plays a crucial role in ribosome biogenesis and related disorders:

  • Component of the small subunit (SSU) processome, the first precursor of the small eukaryotic ribosomal subunit

  • Involved in RNA folding, modifications, and processing during ribosome assembly

  • Implicated in diseases characterized by ribosomal protein insufficiency

  • Used to study Diamond-Blackfan anemia through mouse models

  Research with Rps6 hemizygous mice has shown they develop conditions similar to Diamond-Blackfan anemia, including macrocytic anemia, granulocytopenia, and elevated erythrocyte adenosine deaminase levels . These models provide valuable insights into how ribosomal protein deficiencies affect cellular function and disease development.

• How can RPS6 phosphorylation be used to study neuronal activity and brain function?

  RPS6 phosphorylation serves as an important marker for neuronal activity:

  • Indicates local protein synthesis activation in neurons

  • Responds to various neurotransmitters and growth factors

  • Provides insights into synapse-specific translational regulation

  • Helps monitor downstream effects of neuronal stimulation

  According to search result , "RPS6 phosphorylation is commonly used as a marker for neuronal activity and a readout of mammalian target of rapamycin complex 1 (mTORC1) activity." This makes it valuable for studying neuronal activation patterns, synaptic plasticity, and responses to various stimuli or pharmacological interventions in neuroscience research.