RPS6KA5 Antibody, HRP conjugated

What is RPS6KA5 and why is it important in cellular signaling research?

RPS6KA5 (ribosomal protein S6 kinase alpha-5) is a serine/threonine kinase that functions downstream of both the ERK1/2 and p38 MAPK signaling pathways. It plays critical roles in the phosphorylation of transcription factors including CREB1 and ATF1, as well as in the regulation of RELA, STAT3, and ETV1/ER81. The protein contributes to gene activation through histone phosphorylation and functions significantly in regulating inflammatory genes . RPS6KA5 is primarily nuclear but can also be found in the cytoplasm, making it an important target for studying nuclear-cytoplasmic signaling dynamics . Its involvement in stress responses and mitogen-activated pathways makes it relevant for research in inflammation, cancer, and neurological disorders.

What are the common applications for RPS6KA5 antibody, HRP conjugated?

For protocols requiring higher sensitivity, the HRP conjugation provides enhanced detection capabilities in ELISA applications compared to unconjugated antibodies that require additional detection steps .

How can I validate the specificity of my RPS6KA5 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For RPS6KA5 antibody, consider these methodological approaches:

• Western blot analysis using positive control lysates from cells known to express RPS6KA5 (approximately 90 kDa molecular weight) .

• Include negative controls such as knockout cell lines or siRNA-treated samples.

• Immunoprecipitation followed by mass spectrometry to confirm target identity.

• Peptide blocking experiments using the immunogen peptide to confirm binding specificity.

• Cross-validation with multiple antibodies targeting different epitopes of RPS6KA5.

Immunocytochemistry can also verify subcellular localization, which should show predominantly nuclear staining with some cytoplasmic presence as confirmed in validation images for phospho-specific variants . Always include proper controls and consider that post-translational modifications may affect the observed molecular weight from the theoretical 90 kDa .

How should I design experiments to distinguish between RPS6KA5 phosphorylation states?

Distinguishing between different phosphorylation states of RPS6KA5 requires careful experimental design:

• Select phospho-specific antibodies targeting key regulatory sites (S212, S376, T581, S360, Y449) based on your research question .

• Design time-course experiments following stimulation with known activators (e.g., EGF, UV, stress inducers) to capture dynamic phosphorylation patterns.

• Use phosphatase inhibitors in lysate preparation to preserve phosphorylation status.

• Include parallel Western blots with phospho-specific and total RPS6KA5 antibodies to calculate phosphorylation ratios.

• Consider dual immunofluorescence staining to simultaneously visualize total and phosphorylated RPS6KA5.

For quantitative assessment, phospho-specific ELISA with the HRP-conjugated antibody offers advantages over Western blotting. For instance, ICC staining of MSK1(phospho S376) has been validated in Hela and LO2 cells, showing distinct localization patterns that can be compared with total protein staining . When using inhibitors or activators, include positive controls like samples treated with known MAPK pathway stimulators.

What are the critical considerations for using RPS6KA5 antibody, HRP conjugated in multiplexed assays?

When incorporating RPS6KA5 antibody, HRP conjugated into multiplexed experimental designs:

• Signal separation: HRP generates a colorimetric or chemiluminescent signal that may overlap with other detection systems. Plan your experimental design to avoid signal interference:

  • In multi-color immunofluorescence, ensure proper spectral separation from fluorophores like mFluor Violet 610 SE used with other RPS6KA5 antibodies .

  • For multiplexed Western blots, consider sequential detection with stripping between probes.

• Cross-reactivity assessment: Validate that your RPS6KA5 antibody does not cross-react with related kinases like RSK family members. Most antibodies show reactivity with human, mouse, and rat samples, but specificity should be confirmed .

• Epitope accessibility: When detecting multiple epitopes simultaneously, consider steric hindrance effects that may mask binding sites. Sequential incubation may be necessary for closely positioned epitopes.

• Quantitative normalization: For multiplexed quantitative assays, establish appropriate normalization controls to account for variations in antibody affinity and signal generation between detection systems.

How can I troubleshoot unexpected results when working with phospho-specific RPS6KA5 antibodies?

When encountering unexpected results with phospho-specific RPS6KA5 antibodies:

• High background signal:

  • Increase blocking time/concentration

  • Optimize antibody concentration (perform titration)

  • Ensure sample preparation maintains phosphorylation state with phosphatase inhibitors

  • Consider purification methods that maintain >95% purity as seen with Protein G purified antibodies

• No signal detection:

  • Verify stimulation conditions (duration and concentration)

  • Check phosphatase inhibitor efficacy

  • Confirm storage conditions (-20°C to -80°C for antibodies without azide)

  • Validate antibody on positive control samples where phosphorylation is known to occur

• Multiple bands/non-specific binding:

  • Increase washing stringency

  • Optimize sample preparation to reduce proteolysis

  • Consider antibodies targeting different epitopes (e.g., internal region vs. specific phospho-sites)

  • Verify sample species compatibility with the antibody

• Contradictory localization data:

  • Compare with validated ICC images showing nuclear counterstaining (DAPI)

  • Consider fixation methods (paraformaldehyde has been validated)

  • Verify that stimulation conditions match those reported in literature

What are the key differences between total RPS6KA5 and phospho-specific antibodies in experimental design?

Understanding the distinctions between total and phospho-specific RPS6KA5 antibodies is crucial for proper experimental design:

When designing experiments, total RPS6KA5 antibodies provide information about expression levels, while phospho-specific antibodies reveal functional states. For comprehensive pathway analysis, both should be used in parallel. The available phospho-specific antibodies target sites like S212, S376, T581, S360, and Y449, each with distinct implications for RPS6KA5 activity and downstream effects .

How does fixation method affect the detection of RPS6KA5 in immunocytochemistry?

Fixation methods significantly impact the detection of RPS6KA5 and its phosphorylated forms in immunocytochemistry:

• Paraformaldehyde fixation:

  • Validated for phospho-S376 RPS6KA5 detection in Hela and LO2 cells

  • Preserves protein structure while allowing antibody accessibility

  • Recommended for localization studies showing nuclear predominance with cytoplasmic presence

  • Optimal for co-localization with DAPI nuclear counterstain

• Methanol fixation:

  • May improve detection of certain epitopes but can disrupt phospho-epitopes

  • Generally not preferred for phospho-specific detection

  • Can provide better penetration for some antibodies

• Cross-linking fixatives:

  • May mask epitopes requiring antigen retrieval steps

  • Help preserve cellular architecture for detailed localization studies

When optimizing fixation for RPS6KA5 detection, consider performing parallel experiments with different fixation methods. For phospho-specific detection, paraformaldehyde has shown successful results as evidenced by clear nuclear and cytoplasmic staining patterns in validation images . Always include appropriate controls fixed under identical conditions.

What are the recommended storage conditions for maintaining RPS6KA5 antibody, HRP conjugated activity?

Proper storage is critical for maintaining the activity of RPS6KA5 antibody, HRP conjugated:

• Temperature requirements:

  • Antibodies with azide preservative: Store at 2°C to 8°C

  • Antibodies without azide (including many HRP conjugates): Store at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles which degrade both antibody binding and HRP enzymatic activity

• Buffer considerations:

  • Some preparations are available in 10mM PBS without BSA or azide

  • HRP conjugates may have specific buffer requirements to maintain enzyme activity

  • Check formulation details (e.g., 50mM Sodium Borate for fluorophore conjugates)

• Aliquoting guidelines:

  • Prepare single-use aliquots to avoid repeated freeze-thaw cycles

  • Use sterile conditions when handling to prevent microbial contamination

  • Consider stability data when planning experiment timelines

• Concentration factors:

  • Available at different concentrations (e.g., 1.0 mg/ml or 200 ug/ml)

  • Higher concentration stocks generally have better stability for long-term storage

Following these storage recommendations will help maintain antibody performance throughout your research project timeline and enhance reproducibility between experiments.

How can I quantitatively analyze RPS6KA5 phosphorylation in response to different stimuli?

Quantitative analysis of RPS6KA5 phosphorylation requires rigorous methodological approaches:

• Western blot densitometry:

  • Normalize phospho-RPS6KA5 signal to total RPS6KA5 for accurate phosphorylation ratios

  • Use validated antibody pairs targeting total protein and specific phospho-sites (S212, S376, T581, S360)

  • Include time-course analysis to capture peak phosphorylation periods

  • Apply appropriate statistical analysis to replicate experiments

• ELISA-based quantification:

  • HRP-conjugated antibodies provide direct detection capability

  • Generate standard curves using recombinant phosphorylated proteins

  • Calculate relative phosphorylation levels across experimental conditions

  • Higher throughput than Western blotting for multiple samples

• Image-based quantification:

  • Measure nuclear/cytoplasmic ratios of phospho-RPS6KA5 using immunofluorescence

  • Quantify co-localization with substrates or interacting proteins

  • Use automated image analysis for unbiased assessment

• Multiplex phospho-protein arrays:

  • Analyze RPS6KA5 phosphorylation in context of pathway activation

  • Compare different phosphorylation sites simultaneously

  • Correlate with downstream substrate phosphorylation

When interpreting results, consider the biological context of each phosphorylation site. For example, S376 phosphorylation has distinct functional consequences compared to S212 or T581 phosphorylation .

What controls should I include when studying RPS6KA5 pathway activation?

Robust experimental design for RPS6KA5 pathway studies requires comprehensive controls:

• Positive controls:

  • Cells treated with known activators (e.g., EGF, PMA, anisomycin)

  • Recombinant active RPS6KA5 protein

  • Cell lines with constitutively active upstream kinases

• Negative controls:

  • Untreated/unstimulated cells

  • Cells treated with specific inhibitors of upstream pathways (MEK, p38 inhibitors)

  • siRNA/shRNA knockdown of RPS6KA5

  • Phosphatase-treated samples for phospho-specific detection

• Antibody validation controls:

  • Secondary antibody-only controls

  • Isotype controls (matching the host species and isotype, e.g., Mouse IgG for monoclonal antibodies)

  • Peptide competition assays

  • Cross-species reactivity verification if working with non-human samples

• Experimental condition controls:

  • Time-matched vehicle controls

  • Dose-response curves to establish optimal stimulation conditions

  • Technical replicates to assess method variability

  • Biological replicates to account for cell population heterogeneity

Including these controls allows for confident interpretation of RPS6KA5 pathway activation data and helps troubleshoot unexpected results.

How do different detection methods compare when using RPS6KA5 antibodies?

Different detection methodologies offer distinct advantages when working with RPS6KA5 antibodies:

When selecting a detection method, consider your specific research question. For example, if studying subcellular translocation upon stimulation, immunofluorescence would be most appropriate despite lower quantitative precision. For accurate measurement of phosphorylation levels across many samples, ELISA with HRP-conjugated antibodies provides advantages in throughput and quantification.

What are the recommended approaches for studying RPS6KA5 interactions with its substrates?

Investigating RPS6KA5 interactions with substrates requires specialized methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use validated RPS6KA5 antibodies (non-HRP conjugated) for pull-down experiments

  • Analyze precipitates for known substrates (CREB1, ATF1, histones)

  • Consider crosslinking approaches for transient interactions

  • Include appropriate IgG controls and input samples

• Proximity ligation assay (PLA):

  • Detect in situ protein-protein interactions at single-molecule resolution

  • Several RPS6KA5 antibodies are validated for PLA applications

  • Provides spatial information about interaction sites within cells

  • Quantifiable through image analysis

• Kinase assays:

  • Immunoprecipitate RPS6KA5 using validated antibodies

  • Perform in vitro kinase reactions with purified substrates

  • Use phospho-specific antibodies to detect substrate phosphorylation

  • Include ATP controls and kinase inhibitors as validation

• Bimolecular fluorescence complementation (BiFC):

  • Study direct interactions in living cells

  • Requires genetic fusion constructs rather than antibodies

  • Complements antibody-based approaches for validation

• Chromatin immunoprecipitation (ChIP):

  • Investigate RPS6KA5 association with chromatin

  • Particularly relevant given its role in histone phosphorylation

  • Can be combined with sequencing for genome-wide analysis

These methodologies provide complementary information about RPS6KA5 substrate interactions, with different strengths in detecting stable versus transient interactions or providing spatial information versus biochemical validation.