RPS6KA5 Antibody

What is RPS6KA5/MSK1 and why is it important in cellular research?

RPS6KA5 (ribosomal protein S6 kinase alpha-5), commonly known as MSK1 (Mitogen- and Stress-activated Protein Kinase 1), is a 90 kDa serine/threonine kinase that plays crucial roles in cellular signal transduction. The protein is predominantly found in the nucleus but can also be present in the cytoplasm . It belongs to the protein kinase superfamily and functions as a mediator in various signaling cascades, particularly those involved in stress responses and mitogenic stimulation. RPS6KA5/MSK1 is important in research because it phosphorylates multiple substrates including transcription factors and histones, directly affecting gene expression in response to cellular signals. Studies involving RPS6KA5 antibodies are essential for understanding stress signaling, inflammation processes, and various pathological conditions where these pathways may be dysregulated .

What applications are RPS6KA5 antibodies typically used for in laboratory research?

RPS6KA5 antibodies are versatile tools used across multiple experimental techniques in molecular and cellular biology research. Based on validated applications, these antibodies can be effectively used in:

When designing experiments, it's important to optimize these recommended dilutions for your specific research conditions and sample types .

How should RPS6KA5 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of RPS6KA5 antibodies are critical for maintaining their specificity and sensitivity. The following guidelines should be followed:

For antibodies with preservatives (containing sodium azide):

• Store at 2-8°C for short-term use (up to one month)

• Avoid repeated freeze-thaw cycles as this can degrade antibody quality and reduce binding efficiency

For antibodies without preservatives (azide-free formulations):

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

• Aliquot upon first thaw to minimize freeze-thaw cycles

For conjugated antibodies (e.g., DyLight 488):

• Store at 4°C in the dark to prevent photobleaching of the fluorophore

• Protect from prolonged light exposure during experimental procedures

Most RPS6KA5 antibodies are supplied in buffered solutions (PBS, sodium borate) with stabilizers like glycerol . The presence of 50% glycerol in many formulations helps prevent freezing at -20°C and maintains antibody stability . Always refer to the specific manufacturer's recommendations for the particular antibody you're using, as formulations may vary.

How do phospho-specific RPS6KA5 antibodies differ from total protein antibodies, and when should each be used?

Phospho-specific and total RPS6KA5 antibodies serve different research purposes and provide complementary information about protein activation states:

Phospho-specific RPS6KA5 antibodies (such as phospho S376):

• Recognize RPS6KA5 only when phosphorylated at specific residues (e.g., Serine 376)

• Are generated using synthetic phospho-peptides corresponding to regions surrounding the phosphorylation site

• Should be used when investigating activation status of the kinase, as phosphorylation often correlates with enzymatic activity

• Are ideal for studying signal transduction dynamics, pathway activation, and response to treatments or stimuli

• May show temporal changes in signal intensity following cellular stimulation

Total RPS6KA5 antibodies:

For comprehensive signaling studies, researchers often use both antibody types in parallel. The ratio of phosphorylated to total protein provides insight into the relative activation state normalized to expression levels. When investigating novel treatments or conditions, examining both phosphorylation status and total protein levels helps distinguish between effects on protein activation versus effects on protein expression or stability .

What controls and validation steps should be included when using RPS6KA5 antibodies in experimental work?

Rigorous validation is essential for generating reliable data with RPS6KA5 antibodies. Consider the following controls and validation approaches:

Essential controls:

• Positive control: Lysates from cell lines known to express RPS6KA5 (e.g., HeLa, LO2)

• Negative control: Samples where the protein is absent or knocked down (siRNA/shRNA treatment)

• Isotype control: For flow cytometry and immunostaining to control for non-specific binding

• Primary antibody omission: To evaluate secondary antibody specificity and background

• Blocking peptide control: Competition assays with the immunogenic peptide can confirm specificity

Validation steps:

• Molecular weight verification: Confirm the detected band appears at the expected molecular weight (approximately 90-111 kDa for RPS6KA5)

• Phosphatase treatment: For phospho-specific antibodies, sample treatment with phosphatases should diminish or eliminate signal

• Stimulation experiments: For phospho-antibodies, treatment with known activators (e.g., stress stimuli, growth factors) should increase signal intensity

• Multiple detection methods: Cross-validate findings using different techniques (e.g., Western blot and immunostaining)

• Cross-reactivity assessment: Test the antibody against related proteins (other RSK family members) to ensure specificity

Many manufacturers validate their antibodies through multiple applications. For example, the referenced RPS6KA5 antibodies underwent validation in Western blot, immunocytochemistry, flow cytometry, and protein arrays with appropriate controls to ensure specificity and high affinity .

How can researchers effectively optimize immunostaining protocols for RPS6KA5 detection in different cellular compartments?

RPS6KA5/MSK1 exhibits both nuclear and cytoplasmic localization, making proper optimization of immunostaining protocols critical for accurate subcellular detection:

Fixation optimization:

• Paraformaldehyde (PFA) fixation: Commonly used and preserves most epitopes while maintaining cellular architecture

• Methanol fixation: May provide better access to some nuclear antigens but can distort certain epitopes

• Fixation timing: Over-fixation can mask epitopes; typically 10-20 minutes with 4% PFA is sufficient

Permeabilization considerations:

• Nuclear permeabilization: Since RPS6KA5 is predominantly nuclear, ensure sufficient permeabilization using 0.1-0.5% Triton X-100 or 0.1-0.2% Saponin

• Balanced approach: Excessive permeabilization may reduce cytoplasmic staining while improving nuclear access

Antibody optimization:

• Titration: Test a range of dilutions from 1:50 to 1:200 as recommended by manufacturers

• Incubation time: Longer incubations (overnight at 4°C) may improve specific binding while reducing background

• Blocking optimization: Use 5-10% serum from the species of the secondary antibody along with BSA to minimize non-specific binding

Visualization strategies:

• Co-staining with compartment markers: Use DAPI for nuclear counterstaining as seen in validated images

• Confocal microscopy: For precise subcellular localization, use optical sectioning to discriminate between nuclear and cytoplasmic signals

• Quantitative analysis: Measure nuclear/cytoplasmic ratios of RPS6KA5 staining to assess redistribution under experimental conditions

Research has shown that in unstimulated cells, RPS6KA5 shows predominantly nuclear localization, but stress or mitogenic stimuli can affect this distribution. The immunofluorescence images from validated antibodies confirm nuclear localization with some cytoplasmic presence, as seen in the HeLa cell staining patterns .

What are the key considerations when designing experiments to study RPS6KA5 phosphorylation dynamics?

Studying RPS6KA5 phosphorylation dynamics requires careful experimental design to capture the often transient nature of phosphorylation events:

Temporal considerations:

• Time course experiments: Include multiple time points (5, 15, 30, 60 minutes) after stimulation to capture phosphorylation kinetics

• Rapid processing: Minimize the time between cell lysis and protein denaturation to preserve phosphorylation status

• Phosphatase inhibitors: Always include comprehensive phosphatase inhibitor cocktails in lysis buffers to prevent ex vivo dephosphorylation

Stimulation protocols:

• Pathway-specific activators: Use appropriate stimuli known to activate RPS6KA5, such as EGF, UV radiation, or inflammatory cytokines

• Concentration gradients: Test multiple concentrations of stimuli to establish dose-response relationships

• Inhibitor pre-treatment: Include pathway inhibitors (e.g., MEK inhibitors, p38 inhibitors) to confirm signaling specificity

Technical approaches:

• Phospho-site specificity: Select antibodies targeting relevant phosphorylation sites (e.g., S376) based on your research question

• Multiple readout methods: Combine Western blotting with immunofluorescence to assess both quantity and subcellular localization of phosphorylated RPS6KA5

• Quantitative analysis: Use densitometry with phospho/total protein ratios rather than absolute phospho-signal intensity

Sample preparation:

• Lysis conditions: Use denaturing lysis buffers (containing SDS) for Western blot to fully solubilize nuclear proteins

• Protein loading: Standardize protein loading (20-50 μg per lane) with careful quantification to enable accurate comparisons

• Multiple biological replicates: Include at least three independent experiments to account for biological variability

The phospho-specific antibodies against Serine 376 of RPS6KA5 have been validated to detect the phosphorylated form in multiple applications, making them suitable for studying activation dynamics in response to various stimuli .

How can researchers troubleshoot common issues when using RPS6KA5 antibodies in Western blotting?

Western blotting with RPS6KA5 antibodies may present challenges due to the protein's molecular weight, nuclear localization, and phosphorylation status. Here are solutions to common issues:

For RPS6KA5 detection in Western blotting, manufacturers recommend using 1:1,000 to 1:2,000 dilutions . The observed molecular weight may vary from the calculated 90 kDa to approximately 111 kDa due to post-translational modifications . Using fresh lysates and ensuring complete solubilization of nuclear proteins is particularly important for this predominantly nuclear protein .

What are the optimal cell models and experimental conditions for studying RPS6KA5 function and regulation?

Selecting appropriate cell models and experimental conditions is crucial for meaningful RPS6KA5 research:

Optimal cell models:

• HeLa cells: Widely used and validated for RPS6KA5 studies, showing good expression and nuclear localization as demonstrated in immunofluorescence images

• LO2 cells: Liver cells used for studying RPS6KA5 in hepatic functions

• Primary neurons: Relevant for studying RPS6KA5's role in neuronal plasticity and neuroprotection

• Immune cells: Macrophages and T cells for studying inflammatory signaling pathways involving RPS6KA5

Stimulation conditions:

• Growth factors: EGF, FGF, or serum for activating ERK-dependent pathways

• Stress inducers: UV radiation, hydrogen peroxide, or anisomycin for p38 MAPK pathway activation

• Inflammatory mediators: LPS, TNF-α, or IL-1β for studying RPS6KA5 in inflammatory responses

• Temporal considerations: Short stimulations (15-30 minutes) for acute phosphorylation; longer treatments (hours) for downstream effects

Inhibitor strategies:

• Pathway inhibitors: MEK inhibitors (U0126, PD98059) or p38 inhibitors (SB203580) to define upstream activators

• Direct RPS6KA5 inhibitors: Small molecules like SB-747651A for specific inhibition

• Genetic approaches: siRNA, shRNA, or CRISPR-Cas9 for specific knockdown/knockout studies

Functional readouts:

• Phosphorylation of downstream targets: CREB, histone H3, or ATF1

• Transcriptional regulation: Reporter assays or RT-qPCR for RPS6KA5-regulated genes

• Chromatin immunoprecipitation: To analyze histone modifications mediated by RPS6KA5 activity

Studies have validated RPS6KA5 antibodies in various cell types including HeLa and LO2, making these reliable models for initial studies . The subcellular localization pattern (predominantly nuclear with some cytoplasmic presence) has been consistently observed across these models, providing confidence in their suitability for RPS6KA5 research .

How can researchers effectively combine RPS6KA5 antibody-based techniques with other methodologies for comprehensive signaling pathway analysis?

Integrating multiple technical approaches provides a more complete understanding of RPS6KA5 function within signaling networks:

Multi-technique integration strategies:

• Antibody-based methods with genetic approaches:

  • Combine Western blotting with RPS6KA5 knockdown/knockout to validate specificity and establish causality

  • Use phospho-specific antibodies in cells expressing mutant RPS6KA5 (phospho-mimetic or phospho-deficient) to understand functional consequences

• Spatial and temporal analysis combination:

  • Pair Western blot quantification with live-cell imaging of fluorescently-tagged RPS6KA5

  • Combine fixed-cell immunofluorescence with time-lapse microscopy to correlate localization with activity

• High-throughput with detailed mechanistic studies:

  • Follow protein array screening with targeted co-immunoprecipitation to confirm specific interactions

  • Use flow cytometry for population-level analysis, then select subpopulations for detailed single-cell immunofluorescence

Data integration frameworks:

• Correlation analysis:

  • Plot phosphorylation levels against functional outcomes (e.g., gene expression, cell behavior)

  • Perform Pearson or Spearman correlation between RPS6KA5 activity and downstream target phosphorylation

• Pathway reconstruction:

  • Use RPS6KA5 antibody data as input for computational pathway modeling

  • Integrate phosphoproteomics with RPS6KA5 immunoprecipitation to identify direct and indirect targets

• Systems-level analysis:

  • Combine RPS6KA5 antibody data with transcriptomics to link kinase activity to gene expression changes

  • Integrate with mass spectrometry to identify novel RPS6KA5 interactors or substrates

Researchers have successfully used RPS6KA5 antibodies across multiple platforms including Western blot, immunocytochemistry, flow cytometry, and protein arrays , demonstrating the feasibility of integrated approaches for studying this kinase in diverse cellular contexts.

What are the critical differences between monoclonal and polyclonal RPS6KA5 antibodies, and how should researchers select between them?

The choice between monoclonal and polyclonal RPS6KA5 antibodies significantly impacts experimental outcomes and should be based on specific research requirements:

The search results show examples of both types: monoclonal antibodies like PCRP-RPS6KA5-1A8 produced in mouse, and polyclonal antibodies like the phospho-S376 specific antibody produced in rabbit .

For phosphorylation studies, consider that the polyclonal phospho-specific antibody was generated using synthetic phospho-peptides corresponding to residues surrounding Ser376 , while the monoclonal was produced against full-length recombinant protein . This difference in immunogen may affect how these antibodies perform in detecting native versus denatured protein conformations.

How can researchers properly interpret contradictory results when using different RPS6KA5 antibodies in the same experiment?

Contradictory results between different RPS6KA5 antibodies are not uncommon and require systematic investigation:

Sources of discrepancies:

• Epitope differences: Antibodies targeting different regions or modifications of RPS6KA5 may yield different results

  • Phospho-specific antibodies vs. total protein antibodies

  • N-terminal vs. C-terminal epitope targeting antibodies

  • Conformation-sensitive epitopes may be affected by experimental conditions

• Technical variations:

  • Different optimal dilutions (1:50-1:200 for immunocytochemistry vs. 1:1,000-1:2,000 for Western blotting)

  • Variations in detection sensitivity between applications

  • Buffer compatibility issues affecting epitope recognition

• Biological factors:

  • RPS6KA5 undergoes multiple phosphorylation events and conformational changes

  • Alternative splicing or post-translational modifications may affect epitope availability

  • Subcellular localization (nuclear vs. cytoplasmic) may impact antibody accessibility

Systematic resolution approach:

• Validation with controls:

  • Use positive controls (HeLa lysates) and negative controls (knockdown samples)

  • Include blocking peptide competition to confirm specificity

  • Verify with orthogonal methods (mass spectrometry)

• Cross-antibody comparison:

  • Create a validation matrix testing multiple antibodies against the same samples

  • Document epitope information and cross-reference with known protein domains

  • Use antibodies from different species to enable co-staining experiments

• Careful data interpretation:

  • Consider that discrepancies may reflect real biological complexity rather than technical artifacts

  • Report results from multiple antibodies rather than selecting only confirming data

  • Acknowledge limitations in the discussion section of publications

When faced with contradictory results, researchers should consider that the phosphorylation status of RPS6KA5 is highly dynamic and context-dependent. The observed molecular weight can range from the calculated 90 kDa to approximately 111 kDa due to post-translational modifications , which may contribute to differential antibody recognition.

What are the key differences between commercially available RPS6KA5 antibodies, and how should researchers select the most appropriate one for their specific application?

The available RPS6KA5 antibodies differ in several important characteristics that impact their suitability for specific research applications:

Selection guidance based on research goals:

• For signaling pathway activation studies:

  • Choose the phospho-specific Anti-MSK1 (phospho S376) to monitor specific activation events

  • Optimal for stress or mitogen stimulation experiments where S376 phosphorylation is indicative of activation

• For protein localization studies:

  • Consider the directly conjugated MSK1/RPS6KA5 DyLight 488 for direct visualization

  • Alternatively, use unconjugated antibodies with appropriate secondary antibodies for signal amplification

• For quantitative analyses:

  • Select monoclonal antibodies for consistent reproducibility across experiments

  • Use both phospho-specific and total protein antibodies to calculate activation ratios

• For multiplex experiments:

  • Choose antibodies from different host species to allow co-staining

  • Consider directly conjugated antibodies to eliminate secondary antibody cross-reactivity

• For specialized applications:

  • For flow cytometry: The validated monoclonal antibody has demonstrated specific staining

  • For protein arrays: The monoclonal antibody has shown high specificity in array formats

When selecting antibodies, also consider sample type compatibility. The search results indicate species reactivity differences, with some antibodies validated for both human and rat samples , while others are primarily validated for human samples .

How do different sample preparation methods affect the performance of RPS6KA5 antibodies in various experimental techniques?

Sample preparation significantly impacts RPS6KA5 antibody performance across different experimental platforms:

For Western blotting:

• Lysis buffer composition:

  • RIPA buffer: Provides good solubilization but may affect some epitopes

  • NP-40/Triton X-100 buffers: Milder detergents that preserve protein-protein interactions

  • SDS-based buffers: Most effective for nuclear protein extraction but denature proteins completely

  • Recommended: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate) to preserve phosphorylation status

• Protein denaturation:

  • Standard denaturation (95°C, 5 min) may cause aggregation of high molecular weight proteins

  • Recommended: Denature at 70°C for 10 minutes to minimize aggregation while ensuring epitope exposure

  • For phospho-specific detection , avoid excessive heating which can cause dephosphorylation

For immunocytochemistry/immunofluorescence:

• Fixation methods:

  • Paraformaldehyde (4%, 10-15 min): Preserves morphology while maintaining most epitopes

  • Methanol fixation: May improve access to some nuclear epitopes but can disrupt membrane structures

  • Fixation duration: Critical for balanced preservation of structure and epitope accessibility

• Permeabilization factors:

  • Triton X-100 (0.1-0.5%): Effective for nuclear proteins like RPS6KA5

  • Saponin (0.1-0.2%): Gentler permeabilization that better preserves cytoplasmic epitopes

  • Timing: Brief permeabilization (5-10 min) often sufficient for nuclear proteins

For flow cytometry:

• Cell preparation:

  • PFA fixation followed by gentle permeabilization recommended

  • Single-cell suspensions critical for accurate analysis

  • Careful titration of antibody (1-2 μg/million cells) to minimize background

For immunoprecipitation:

• Lysis conditions:

  • Non-denaturing buffers (NP-40, Triton X-100) preserve protein-protein interactions

  • Pre-clearing lysate reduces non-specific binding

  • Antibody amounts need optimization (typically 2-5 μg per mg protein)

Images from validated immunofluorescence experiments show that proper fixation and permeabilization enable clear visualization of the predominantly nuclear localization of RPS6KA5, with some cytoplasmic presence . For optimal results, researchers should carefully match sample preparation methods to the specific antibody and application being used.

What novel or emerging applications of RPS6KA5 antibodies are being developed in current research?

RPS6KA5 antibodies are increasingly being utilized in innovative research applications that extend beyond traditional techniques:

Advanced imaging approaches:

• Super-resolution microscopy: Using highly specific RPS6KA5 antibodies to visualize nanoscale distribution patterns within nuclear subdomains

• Live-cell proximity labeling: Combining RPS6KA5 antibody fragments with enzyme tags for spatiotemporal tracking of kinase activity

• Multiplexed imaging: Simultaneous detection of RPS6KA5 with multiple signaling components using spectral unmixing and antibody panels

Single-cell analysis technologies:

• Mass cytometry (CyTOF): Metal-conjugated RPS6KA5 antibodies for high-dimensional analysis of signaling states at single-cell resolution

• Single-cell Western blotting: Microfluidic platforms using RPS6KA5 antibodies to analyze protein expression heterogeneity

• Spatial proteomics: In situ detection of RPS6KA5 and its substrates with subcellular resolution using multiplexed antibody staining

Therapeutic and diagnostic developments:

• Antibody-drug conjugates: Using RPS6KA5 antibodies to deliver targeted therapies to cells with aberrant kinase expression

• Companion diagnostics: RPS6KA5 phosphorylation status as a biomarker for response to targeted therapies

• Nanobody and aptamer derivatives: Developing smaller binding molecules based on RPS6KA5 antibody epitope mapping

High-throughput applications:

• Protein arrays: As validated in the search results , RPS6KA5 antibodies are being used in protein array formats for interaction screening

• Automated tissue analysis: Machine learning algorithms combined with RPS6KA5 immunohistochemistry for pattern recognition in tissue samples

• CRISPR screens: Using RPS6KA5 antibodies as readouts for large-scale genetic perturbation studies

These emerging applications leverage the high specificity of RPS6KA5 antibodies, particularly monoclonal antibodies , which provide consistent results across different experimental platforms. The development of directly conjugated antibodies, such as the DyLight 488 conjugate , further enables novel multiparameter analyses in both imaging and cytometry applications.

What are the most critical factors researchers should consider when designing experiments with RPS6KA5 antibodies?

The successful use of RPS6KA5 antibodies in research requires careful consideration of several critical factors:

• Antibody selection matching research question:

  • For activation studies: Use phospho-specific antibodies like Anti-MSK1 (phospho S376)

  • For expression studies: Use antibodies recognizing total protein regardless of phosphorylation state

  • For subcellular localization: Consider directly conjugated antibodies or those validated for immunofluorescence

• Experimental controls:

  • Include positive controls (cell lines with known RPS6KA5 expression like HeLa)

  • Implement negative controls (isotype controls, primary antibody omission)

  • For phospho-antibodies, include samples with relevant pathway activation and inhibition

• Technical optimization:

  • Adjust antibody concentrations based on application (1:50-1:200 for ICC/IHC; 1:1,000-1:2,000 for WB)

  • Select appropriate sample preparation methods based on subcellular localization (nuclear/cytoplasmic)

  • Include proper blocking to minimize background (5-10% serum or BSA)

• Biological context:

  • Consider cell type-specific expression patterns and signaling dynamics

  • Account for potential cross-reactivity with related kinases

  • Integrate findings with known pathway information for meaningful interpretation

• Reproducibility considerations:

  • Document detailed protocols including antibody catalog numbers and dilutions

  • Record lot numbers to account for potential lot-to-lot variations

  • Validate findings using multiple antibodies or orthogonal approaches when possible