Phospho-RPS6KA5 (S376) Recombinant Monoclonal Antibody

What is RPS6KA5 and why is phosphorylation at S376 significant?

RPS6KA5, also known as ribosomal protein S6 kinase A5 or MSK1, is a protein kinase involved in numerous cellular processes, including cell growth, proliferation, and survival. It plays a crucial role in intracellular signaling pathways that regulate gene expression and cell behavior in response to various stimuli .

Phosphorylation at S376 is a crucial regulatory mechanism that allows cells to respond to extracellular signals and stressors, modulating gene expression and influencing various cellular processes. When this phosphorylation event is dysregulated, it can have significant implications in diseases and conditions related to cell growth, stress responses, and gene expression .

Understanding this phosphorylation event requires careful experimental design, including:

• Appropriate cell stimulation protocols

• Positive and negative controls

• Time-course experiments to capture dynamic phosphorylation changes

• Validation with multiple detection methods

How does this antibody differ from other RPS6KA5 phospho-specific antibodies?

The Phospho-RPS6KA5 (S376) recombinant monoclonal antibody specifically recognizes the phosphorylated serine at position 376, while other phospho-specific antibodies target different phosphorylation sites on RPS6KA5, such as T581 . Each phosphorylation site has distinct biological significance and regulation pathways.

Table 1: Comparison of RPS6KA5 Phospho-Specific Antibodies

Methodologically, researchers should:

• Select the appropriate phospho-specific antibody based on the specific research question

• Use multiple phospho-specific antibodies when studying activation mechanisms

• Include both phospho-specific and total protein antibodies in experiments

What are the typical applications for this antibody?

The Phospho-RPS6KA5 (S376) recombinant monoclonal antibody is primarily used for immunohistochemistry (IHC) at recommended dilutions of 1:50-1:200 . This application is particularly valuable for:

• Visualizing the spatial distribution of phosphorylated RPS6KA5 in tissue samples

• Assessing activation status of RPS6KA5 in different cell types within complex tissues

• Comparing phosphorylation levels between normal and pathological tissue samples

• Correlating phosphorylation status with disease progression or treatment response

For optimal results in IHC applications:

• Use appropriate antigen retrieval methods

• Optimize antibody concentration for each tissue type

• Include positive and negative controls

• Consider counterstaining to provide cellular context

How does RPS6KA5 phosphorylation relate to its kinase activity?

RPS6KA5 (MSK1) phosphorylation status directly affects its kinase activity toward various substrates. Research has shown that RPS6KA5 can phosphorylate ubiquitin at S57, and this activity appears to be dependent on the activation state of RPS6KA5 itself .

Methodologically, when studying RPS6KA5 kinase activity:

• Use in vitro kinase assays with purified components

• Employ Phos-tag™ acrylamide SDS-PAGE to separate phosphorylated and non-phosphorylated forms

• Utilize fingerprint mass-spectrometry to identify specific phosphorylation sites

• Compare wild-type and phospho-mutant versions of RPS6KA5

Table 2: Known Substrates of RPS6KA5

What are the molecular mechanisms regulating S376 phosphorylation?

The phosphorylation of RPS6KA5 at S376 is regulated through complex molecular mechanisms that involve upstream kinases, phosphatases, and scaffolding proteins. The phosphorylation state can be influenced by:

• Growth factor stimulation (e.g., EGF, PDGF)

• Cellular stress (oxidative stress, DNA damage)

• Inflammatory signals

• Energy status of the cell

To study these regulatory mechanisms:

• Use specific inhibitors of upstream kinases

• Employ siRNA/shRNA knockdown of regulatory proteins

• Utilize phosphatase inhibitors to prevent dephosphorylation

• Perform time-course experiments following various stimuli

How reliable are phospho-specific antibodies for detecting transient phosphorylation events?

Detecting transient phosphorylation events is challenging but can be accomplished with careful methodology. The Phospho-RPS6KA5 (S376) recombinant monoclonal antibody is produced by isolating genes responsible for coding this antibody from rabbits that have been previously exposed to a synthesized peptide originating from the human RPS6KA5 protein phosphorylated at S376 .

For reliable detection of transient phosphorylation:

• Use rapid cell lysis techniques with phosphatase inhibitors

• Perform time-course experiments with short intervals

• Consider using phospho-enrichment techniques before western blotting

• Validate results with multiple approaches (e.g., mass spectrometry)

• Include proper controls for antibody specificity

Table 3: Methods for Detecting Transient Phosphorylation Events

What controls should be included when using Phospho-RPS6KA5 (S376) antibody?

When designing experiments using the Phospho-RPS6KA5 (S376) recombinant monoclonal antibody, proper controls are essential for reliable interpretation of results:

• Positive controls:

  • Cell lysates from cells treated with known activators of the pathway

  • Recombinant phosphorylated protein (if available)

  • Tissues known to express phosphorylated RPS6KA5

• Negative controls:

  • Cell lysates treated with phosphatase

  • Samples from RPS6KA5 knockdown/knockout models

  • S376A mutant-expressing cells (where serine is replaced with alanine)

  • Use of blocking peptides containing the phosphorylated epitope

• Specificity controls:

  • Parallel detection with total RPS6KA5 antibody

  • Competitive blocking with the immunogenic phosphopeptide

  • Comparison with other detection methods

How can researchers optimize immunohistochemistry protocols with this antibody?

Optimizing IHC protocols for the Phospho-RPS6KA5 (S376) recombinant monoclonal antibody requires careful attention to several key factors:

• Tissue preparation:

  • Ensure proper fixation (typically 10% neutral buffered formalin)

  • Optimize fixation time to preserve phospho-epitopes

  • Use freshly cut sections when possible

• Antigen retrieval:

  • Test multiple retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer conditions (citrate, EDTA, Tris)

  • Determine optimal retrieval time and temperature

• Antibody dilution:

  • Perform dilution series (starting with 1:50-1:200 as recommended)

  • Include positive and negative controls at each dilution

  • Optimize primary antibody incubation time and temperature

• Detection system:

  • Select appropriate secondary antibody system

  • Consider signal amplification for low-abundance phospho-proteins

  • Optimize DAB development time

• Validation:

  • Perform peptide competition assays

  • Compare with western blot results from the same samples

  • Include phosphatase-treated sections as controls

What considerations are important when validating antibody specificity?

Validation of the Phospho-RPS6KA5 (S376) recombinant monoclonal antibody's specificity is crucial for generating reliable research data. The manufacturer typically validates antibodies through ELISA and IHC, confirming their capability to interact effectively with the human RPS6KA5 protein phosphorylated at S376 .

Researchers should implement additional validation steps:

• Genetic approaches:

  • Use RPS6KA5 knockout/knockdown models

  • Express phospho-mutant versions (S376A)

  • Compare wild-type vs. kinase-inactive mutants

• Pharmacological approaches:

  • Use specific inhibitors of upstream pathways

  • Treat samples with phosphatases

  • Stimulate cells with activators of the pathway

• Analytical approaches:

  • Perform phosphopeptide mapping

  • Use mass spectrometry to confirm specificity

  • Compare results with other phospho-specific antibodies

• Cross-reactivity assessment:

  • Test against closely related family members

  • Evaluate species cross-reactivity

  • Check for non-specific binding in various cell types

How can researchers address weak or inconsistent phospho-RPS6KA5 (S376) signals?

Weak or inconsistent signals when using the Phospho-RPS6KA5 (S376) recombinant monoclonal antibody can result from various factors. Methodological approaches to address these issues include:

• Sample preparation issues:

  • Ensure rapid sample collection and processing

  • Use fresh phosphatase inhibitors in lysis buffers

  • Avoid freeze-thaw cycles of protein samples

  • Consider using phospho-protein enrichment methods

• Technical considerations:

  • Optimize antibody concentration (try higher concentrations within the 1:50-1:200 range)

  • Increase protein loading amount

  • Extend primary antibody incubation time

  • Try different detection systems with higher sensitivity

• Biological factors:

  • Confirm appropriate stimulation conditions

  • Consider the half-life of the phosphorylation event

  • Check for cell-type specific differences in phosphorylation

  • Evaluate the abundance of the protein in your model system

• Antibody-related issues:

  • Verify antibody storage conditions

  • Check antibody lot-to-lot variation

  • Consider testing alternative phospho-antibodies

  • Validate with recombinant phosphorylated standards

What factors might cause discrepancies between different phospho-specific antibodies?

When researchers observe discrepancies between different phospho-specific antibodies targeting RPS6KA5, several factors may contribute:

• Epitope accessibility differences:

  • Conformational changes affecting epitope exposure

  • Protein interactions masking specific phospho-sites

  • Post-fixation alterations to epitope structure

• Antibody characteristics:

  • Differences in affinity and specificity

  • Clonality differences (monoclonal vs. polyclonal)

  • Cross-reactivity with similar phospho-motifs

  • Host species differences affecting background

• Biological considerations:

  • Differential regulation of various phosphorylation sites

  • Site-specific phosphorylation kinetics

  • Subcellular localization changes upon phosphorylation

  • Context-dependent phosphorylation patterns

• Technical variations:

  • Different optimal conditions for each antibody

  • Variations in sample preparation methods

  • Detection system sensitivity differences

  • Lot-to-lot variability in antibody production

To resolve discrepancies, researchers should:

• Perform parallel validations of all antibodies

• Use multiple detection methods

• Consider phospho-site interactions (priming effects)

• Employ mass spectrometry for unbiased confirmation

How can quantitative analysis of RPS6KA5 phosphorylation be optimized?

Quantitative analysis of RPS6KA5 phosphorylation requires careful experimental design and appropriate analytical methods:

• Sample preparation for quantification:

  • Use standardized lysate preparation methods

  • Include calibration standards when possible

  • Process all samples simultaneously

  • Maintain consistent protein loading across samples

• Western blot quantification:

  • Use internal loading controls

  • Calculate phospho-to-total protein ratios

  • Ensure signal is within linear detection range

  • Use digital image analysis software

• Immunohistochemistry quantification:

  • Employ digital pathology systems

  • Use H-score or other semi-quantitative methods

  • Analyze multiple fields per sample

  • Include staining intensity and percent positive cells

• Advanced quantification methods:

  • Consider ELISA-based quantification

  • Utilize phospho-flow cytometry for single-cell analysis

  • Implement multiple reaction monitoring mass spectrometry

  • Use proximity ligation assays for in situ quantification

Table 4: Comparison of Quantification Methods for Phospho-RPS6KA5

How does RPS6KA5 phosphorylation contribute to substrate specificity?

Research indicates that RPS6KA5 phosphorylation status affects its substrate specificity. For example, studies have shown that RPS6KA5 can phosphorylate ubiquitin at S57, and this activity might be regulated by the phosphorylation status of RPS6KA5 itself .

To investigate how RPS6KA5 phosphorylation influences substrate specificity:

• Use constitutively active and phospho-mimetic mutants

• Perform in vitro kinase assays with purified components

• Conduct phosphoproteomics following manipulation of RPS6KA5 phosphorylation

• Analyze structural changes upon phosphorylation using biophysical methods

Advanced research indicates that kinase recruitment is essential for ubiquitin phosphorylation, suggesting that the phosphorylation motif surrounding S57 demonstrates relatively poor peptide specificity for RPS6KA5 . This finding highlights the complexity of substrate recognition beyond simple consensus sequences.

What is the relationship between RPS6KA5 phosphorylation and cellular localization?

RPS6KA5 has been found to localize in both the cytoplasm and nucleus , and its phosphorylation status may influence this distribution. Research methodologies to investigate this relationship include:

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Analyze phospho-RPS6KA5 levels in each fraction

  • Compare different phosphorylation sites

• Live-cell imaging:

  • Generate fluorescently tagged RPS6KA5

  • Create phospho-mimetic and phospho-dead mutants

  • Monitor localization changes upon stimulation

• Immunofluorescence microscopy:

  • Co-stain with phospho-RPS6KA5 (S376) and total RPS6KA5 antibodies

  • Analyze colocalization with nuclear markers

  • Quantify nuclear-to-cytoplasmic ratios

• Biochemical approaches:

  • Analyze nuclear import/export sequences

  • Investigate interaction with transport proteins

  • Assess effects of phosphorylation on protein-protein interactions

How can phospho-RPS6KA5 (S376) be integrated into multi-parameter analyses?

Integrating phospho-RPS6KA5 (S376) analysis into multi-parameter studies provides a more comprehensive understanding of signaling networks. Methodological approaches include:

• Multiplexed immunoassays:

  • Combine phospho-RPS6KA5 (S376) with other pathway components

  • Use different fluorophores or chromogens

  • Analyze spatial relationships between markers

• Systems biology approaches:

  • Correlate phospho-RPS6KA5 (S376) with transcriptomic data

  • Model signaling networks incorporating phosphorylation data

  • Integrate with patient outcome or phenotypic data

• Multi-omics integration:

  • Combine phosphoproteomics, transcriptomics, and metabolomics

  • Perform pathway enrichment analyses

  • Identify regulatory relationships between pathways

• Single-cell analyses:

  • Use mass cytometry (CyTOF) for multiple phospho-proteins

  • Perform spatial proteomics in tissue sections

  • Analyze cell-to-cell variability in phosphorylation patterns