RPS6KA2 Antibody, HRP conjugated

What is RPS6KA2 and why is it important in cellular signaling?

RPS6KA2 (also known as RSK3, p90-RSK3, or S6K-alpha2) belongs to the RSK family of serine/threonine kinases. It contains two non-identical kinase catalytic domains and phosphorylates various substrates, including members of the mitogen-activated protein kinase (MAPK) signaling pathway . The activity of this protein has been implicated in controlling cell growth and differentiation, making it a significant target for research in cellular biology and potential disease mechanisms . RPS6KA2 has multiple isoforms resulting from alternative splicing, each with potentially different functional roles in various tissues. Its involvement in fundamental cellular processes makes it a critical protein to study in both normal physiological and pathological conditions.

What are the recommended storage conditions for maintaining HRP-conjugated RPS6KA2 antibody activity?

HRP-conjugated RPS6KA2 antibodies should be stored at -20°C to maintain optimal activity . It is critical to avoid repeated freeze-thaw cycles as these can degrade both the antibody and the conjugated enzyme activity . Most manufacturers recommend storage in a buffer containing stabilizers such as PBS with 0.01% thiomersal and 50% glycerol at pH 7.3 . For working solutions, storage at 4°C for up to one week is generally acceptable, but longer-term storage should return to -20°C. When handling the antibody, avoid exposure to strong light and contamination with microorganisms or chemicals that could degrade either the antibody or HRP enzyme. Proper aliquoting upon receiving the antibody can minimize the number of freeze-thaw cycles and extend the functional lifespan of the reagent significantly.

What are the optimal dilution ratios for different applications of HRP-conjugated RPS6KA2 antibodies?

The optimal dilution ratios for HRP-conjugated RPS6KA2 antibodies vary significantly depending on the specific application. Based on validated experimental data, the following dilution ranges are recommended:

When working with a new lot or source of antibody, it is advisable to perform a dilution series to determine the optimal concentration for your specific experimental conditions. Factors such as protein abundance, sample type, and detection method will influence the ideal dilution.

How should positive and negative controls be designed for RPS6KA2 antibody experiments?

Proper experimental design for RPS6KA2 antibody validation requires thoughtful selection of controls. For positive controls, cell lines known to express RPS6KA2 should be used, such as human cell lines from various tissues as demonstrated in validated Western blot analyses . Recombinant RPS6KA2 protein can also serve as an excellent positive control, particularly when using the same domain targeted by the antibody (e.g., AA 228-395 or AA 415-734) .

For negative controls, consider the following approaches:

• Cell lines with confirmed low or no expression of RPS6KA2

• Samples treated with RPS6KA2-specific siRNA for knockdown verification

• Absorption controls where the antibody is pre-incubated with the immunizing peptide

• Isotype controls to rule out non-specific binding

For immunohistochemistry applications, include tissues known to lack RPS6KA2 expression or substitute the primary antibody with non-immune serum from the same species. Ideally, all experiments should incorporate both types of controls to ensure the specificity and sensitivity of the detection system.

What is the recommended protocol for using HRP-conjugated RPS6KA2 antibody in Western blotting?

The following optimized protocol is based on validated experimental data for using HRP-conjugated RPS6KA2 antibody in Western blotting:

• Sample preparation: Extract proteins from cells/tissues using appropriate lysis buffer containing protease inhibitors

• Protein quantification: Use Bradford or BCA assay to normalize loading (25μg per lane recommended)

• SDS-PAGE: Separate proteins using 8-10% gels (optimal for 83kDa RPS6KA2 protein)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block with 3% nonfat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute HRP-conjugated RPS6KA2 antibody at 1:1000 in blocking buffer; incubate overnight at 4°C

• Washing: Wash membrane 3×5 minutes with TBST

• Detection: Apply ECL substrate directly (no secondary antibody needed) and visualize

• Exposure: Optimize exposure time based on signal strength (30 seconds as starting point)

For maximum sensitivity and reproducibility, include a loading control antibody (such as β-actin or GAPDH) in a different channel if using a digital imaging system, or on a separate blot if using film detection.

How can researchers minimize background signal when using HRP-conjugated RPS6KA2 antibodies?

Excessive background is a common challenge when working with HRP-conjugated antibodies. Several methodological approaches can minimize this issue:

• Optimize blocking conditions: Test different blocking agents (BSA, casein, commercial blockers) at various concentrations and incubation times. The validated protocol recommends 3% nonfat dry milk in TBST .

• Increase washing stringency: More frequent and longer washing steps with TBST can significantly reduce background. Consider adding 0.05-0.1% Tween-20 to washing buffers to reduce non-specific binding.

• Dilution optimization: Higher dilutions of the HRP-conjugated antibody often reduce background while maintaining specific signal. Perform a dilution series to determine the optimal concentration.

• Sample preparation: Ensure complete lysis and removal of cellular debris through centrifugation. Consider pre-clearing lysates with Protein A/G beads.

• Reduce detection sensitivity: If using an extremely sensitive ECL substrate, consider switching to a less sensitive version that won't amplify background signals.

• Antigen retrieval modification: For IHC/ICC applications, optimize antigen retrieval methods as overheating can increase non-specific binding.

These methodological adjustments should be tested systematically to determine which combination provides the optimal signal-to-noise ratio for your specific experimental system.

What are common causes of false negative results when using RPS6KA2 antibodies and how can they be addressed?

False negative results can occur despite the presence of the RPS6KA2 protein. Common causes and their methodological solutions include:

• Insufficient antigen availability: Ensure proper antigen retrieval for fixed tissues/cells. For IHC, microwave antigen retrieval with 10 mM PBS buffer pH 7.2 is recommended .

• Protein degradation: Use fresh samples or proper preservation methods with protease inhibitors. Store proteins at appropriate temperatures and avoid repeated freeze-thaw cycles.

• Ineffective transfer in Western blotting: Verify transfer efficiency using reversible staining methods like Ponceau S before blocking.

• Incorrect epitope targeting: The HRP-conjugated RPS6KA2 antibody targets specific amino acid sequences (e.g., AA 228-395) . If your protein variant lacks this region due to splicing or post-translational modifications, consider antibodies targeting different epitopes.

• Detection system limitations: HRP activity can be inhibited by sodium azide or strong reducing agents in buffers. Ensure compatibility of all reagents.

• Excessive dilution: If signal is absent, try using the antibody at a lower dilution (e.g., 1:500 instead of 1:2000) .

When troubleshooting, implement methodical changes to identify the specific cause rather than changing multiple variables simultaneously.

How do researchers resolve inconsistent results between different applications of RPS6KA2 antibodies?

Inconsistencies between applications (e.g., positive Western blot but negative IHC) are not uncommon and can be resolved through methodological refinement:

• Application-specific optimization: Each technique exposes different epitopes. The HRP-conjugated RPS6KA2 antibody may require different conditions for optimal performance in Western blotting versus immunohistochemistry .

• Protocol verification: Confirm that recommended dilutions are application-specific. For example, Western blotting typically uses 1:500-1:2000, while IHC may require 1:100-1:200 .

• Epitope accessibility analysis: The AA 228-395 region targeted by some RPS6KA2 antibodies may be accessible in denatured proteins (Western blot) but masked in fixed tissues (IHC). Consider epitope-specific optimization.

• Cross-technique validation: Use multiple detection methods for the same sample. If protein is detected by mass spectrometry but not antibody-based methods, antibody specificity may be the issue.

• Sample preparation standardization: Develop consistent protocols for sample handling across all experiments to reduce variability.

• Buffer system compatibility: Ensure that buffers and reagents are compatible with both the antibody and the HRP enzyme conjugate across different applications.

Systematic testing and detailed record-keeping of experimental conditions will help identify the source of inconsistencies and establish reliable protocols for each application.

How can phospho-specific RPS6KA2 antibodies complement HRP-conjugated total RPS6KA2 antibodies in signaling research?

Combining phospho-specific and total RPS6KA2 antibodies provides a powerful approach for dissecting signaling dynamics. While HRP-conjugated total RPS6KA2 antibodies detect the protein regardless of phosphorylation status , phospho-specific antibodies can reveal activation states of the kinase. This methodological pairing enables researchers to:

• Calculate activation ratios: By normalizing phospho-RPS6KA2 signal to total RPS6KA2, researchers can quantify the proportion of activated kinase independent of expression level changes.

• Track signaling kinetics: Using time-course experiments with both antibody types allows monitoring of both rapid phosphorylation changes and slower protein expression alterations.

• Identify regulatory mechanisms: Differential changes between phosphorylation and total protein can reveal whether a treatment affects RPS6KA2 activity, stability, or expression.

• Map pathway cross-talk: By correlating RPS6KA2 phosphorylation with other MAPK pathway components, researchers can determine signaling hierarchy and feedback mechanisms.

When designing such experiments, it's crucial to ensure that the epitopes recognized by phospho-specific and total antibodies don't interfere with each other if performing simultaneous detection. Sequential detection protocols using stripping and reprobing can circumvent this limitation but require careful validation.

What methodologies integrate RPS6KA2 antibody detection with functional kinase activity assays?

Integrating antibody detection with functional kinase assays provides comprehensive insight into both RPS6KA2 presence and activity. Advanced methodological approaches include:

• Immunoprecipitation kinase assays: Use non-conjugated RPS6KA2 antibodies to immunoprecipitate the kinase, then assess activity through in vitro kinase assays with known substrates and detection of phosphorylation by radiometric or non-radiometric methods.

• Proximity ligation assays (PLA): Combine RPS6KA2 antibodies with antibodies against known interaction partners or substrates to visualize protein-protein interactions in situ, providing spatial information about kinase function .

• Phospho-flow cytometry: Utilize fixed and permeabilized cells stained with fluorophore-conjugated RPS6KA2 antibodies alongside phospho-specific antibodies to correlate expression with activity at single-cell resolution.

• FRET-based biosensors: Engineer fluorescent biosensors that change conformation upon phosphorylation by RPS6KA2, allowing real-time monitoring of kinase activity in living cells.

• Multiplex Western blotting: Simultaneously detect total RPS6KA2, phospho-RPS6KA2, and phosphorylated downstream targets to correlate expression, activation, and functional output.

These integrated approaches bridge the gap between detecting the kinase and understanding its functional significance in complex signaling networks, offering deeper mechanistic insights than either approach alone.

How can HRP-conjugated RPS6KA2 antibodies be adapted for high-throughput screening applications?

Adapting HRP-conjugated RPS6KA2 antibodies for high-throughput screening requires methodological modifications to increase throughput while maintaining specificity and sensitivity:

• Microplate-based ELISA optimization:

  • Use 384 or 1536-well formats to maximize sample processing

  • Adapt antibody dilutions (typically 1:1000-1:5000) to minimize reagent usage while maintaining signal

  • Implement automated liquid handling systems for consistent reagent dispensing

• Multiplex bead-based assays:

  • Couple RPS6KA2 capture antibodies to spectrally distinct microspheres

  • Use HRP-conjugated RPS6KA2 antibodies as detection reagents

  • Enable simultaneous measurement of multiple analytes by focusing on non-overlapping epitopes

• Automated Western blotting systems:

  • Utilize capillary-based protein separation systems that increase throughput

  • Optimize blocking and antibody incubation times for the HRP-conjugated format

  • Implement automated image analysis for consistent quantification

• High-content imaging approaches:

  • Adapt RPS6KA2 immunocytochemistry protocols for automated microscopy

  • Optimize dilutions (1:50-1:200) for cellular imaging applications

  • Develop computational workflows for image segmentation and quantification

• Quality control considerations:

  • Implement routine calibration curves with recombinant standards

  • Include intra-plate controls to account for processing variability

  • Validate hit selection algorithms using known modulators of RPS6KA2

These adaptations enable screening of thousands of conditions while maintaining the specific detection capabilities of HRP-conjugated RPS6KA2 antibodies.

How should researchers interpret variations in RPS6KA2 molecular weight across different experimental systems?

While the calculated molecular weight of RPS6KA2 is 83kDa , researchers often observe variations in apparent molecular weight. These variations require thoughtful interpretation:

• Post-translational modifications: Phosphorylation of RPS6KA2 at multiple sites can significantly alter migration patterns. The protein contains numerous serine/threonine phosphorylation sites that are dynamically regulated during signaling events.

• Alternative splicing: RPS6KA2 exhibits multiple splice variants that result in isoforms of different sizes . When interpreting Western blot results, consider whether your antibody (e.g., targeting AA 228-395 or AA 415-734 ) recognizes all or specific isoforms.

• Species differences: Human RPS6KA2 may migrate differently than mouse or rat orthologs. Cross-species comparisons should account for sequence variations that might affect both antibody recognition and protein migration.

• Technical factors: Electrophoresis conditions, including gel percentage, buffer systems, and running conditions can influence apparent molecular weight. Standardize these parameters when making comparisons across experiments.

• Sample preparation effects: Heat treatment duration, reducing agent concentration, and lysis conditions can all affect protein conformation and migration patterns.

When reporting RPS6KA2 detection, always specify both the expected and observed molecular weights, along with experimental conditions that might influence migration patterns. This methodological transparency facilitates accurate cross-study comparisons.

What are the critical considerations when interpreting RPS6KA2 expression patterns across different tissues and cell types?

Interpreting RPS6KA2 expression patterns requires careful consideration of several key factors:

How does RPS6KA2 detection data complement other approaches in understanding MAPK pathway dynamics?

RPS6KA2 detection data provides valuable insights when integrated with other MAPK pathway analyses:

• Hierarchical signaling reconstruction: RPS6KA2, as a downstream effector of ERK1/2, can be analyzed alongside upstream kinases (RAF, MEK) and downstream substrates to map activation sequences within the pathway. This multi-level detection approach reveals potential rate-limiting steps and feedback mechanisms.

• Temporal resolution of signaling: Combining RPS6KA2 detection with time-course analyses of other MAPK components helps distinguish between immediate-early and delayed responses. This temporal dimension is crucial for understanding signal propagation and termination.

• Spatial distribution analysis: Immunofluorescence studies of RPS6KA2 localization complement data on other MAPK components to reveal compartment-specific signaling events. Subcellular fractionation followed by Western blotting can provide quantitative support for localization data.

• Isoform-specific functions: Detection of specific RPS6KA2 isoforms using epitope-specific antibodies allows researchers to determine whether particular variants have specialized roles within the MAPK pathway.

• Multi-pathway integration: RPS6KA2 participates in cross-talk with other signaling pathways. Simultaneous detection of components from multiple pathways (e.g., PI3K/AKT, JAK/STAT) alongside RPS6KA2 reveals integration points and conditional dependencies.

• Functional validation: RPS6KA2 antibody data should be integrated with functional readouts (e.g., reporter assays, phenotypic changes) to connect molecular detection with biological outcomes.

This integrative approach transforms RPS6KA2 detection from a single data point into a component of comprehensive pathway understanding, enhancing the biological significance of experimental findings.

How might advances in multiplexed antibody detection systems enhance RPS6KA2 signaling research?

Emerging multiplexed detection technologies offer new opportunities for comprehensive analysis of RPS6KA2 within signaling networks:

• Mass cytometry (CyTOF) integration: Adaptation of RPS6KA2 antibodies for metal-conjugation rather than HRP would enable simultaneous detection of dozens of other signaling proteins at single-cell resolution, revealing population heterogeneity in RPS6KA2 signaling responses.

• Digital spatial profiling: This technology would allow spatial mapping of RPS6KA2 expression and activation in tissue contexts alongside numerous other proteins, providing unprecedented insight into microenvironmental regulation of MAPK signaling.

• Single-cell Western blotting: These emerging platforms could detect RPS6KA2 alongside other pathway components in individual cells, revealing cell-to-cell variability that is masked in bulk analyses.

• Sequential multiplexed immunofluorescence: Methods for iterative staining, imaging, and bleaching could locate RPS6KA2 in spatial relationship to dozens of other proteins within the same tissue section.

• Antibody-based proximity labeling: Conjugating RPS6KA2 antibodies to enzymes that catalyze proximity-dependent labeling would enable identification of transient interaction partners in native contexts.

These technological advances will transform RPS6KA2 research from targeted studies to comprehensive network analyses, revealing emergent properties of signaling systems that cannot be discerned from traditional single-protein investigations.

What methodological approaches can integrate RPS6KA2 antibody data with genomic and proteomic datasets?

Integrating RPS6KA2 antibody data with multi-omic datasets requires sophisticated methodological approaches:

• Correlation with transcriptomic data: Compare protein levels detected by RPS6KA2 antibodies with mRNA expression data to identify post-transcriptional regulation mechanisms. Discrepancies between protein and mRNA levels may reveal regulatory control points.

• Phosphoproteomic integration: Correlate total RPS6KA2 levels detected by HRP-conjugated antibodies with phosphopeptide abundance from mass spectrometry to quantify the relationship between expression and activation state across conditions.

• ChIP-seq correlation: Combine RPS6KA2 protein detection with chromatin immunoprecipitation sequencing data for downstream transcription factors to establish functional consequences of kinase activation.

• Protein interaction network mapping: Use RPS6KA2 antibody data as anchor points for interpreting protein-protein interaction networks derived from affinity purification-mass spectrometry or proximity labeling approaches.

• Multi-omic data visualization: Develop computational approaches to visualize RPS6KA2 antibody data within the context of multi-omic datasets, enabling intuitive interpretation of complex relationships.

• Machine learning applications: Train predictive models using RPS6KA2 antibody data combined with multi-omic datasets to identify patterns and relationships not apparent through conventional analysis.

These integrative approaches transform isolated antibody-based measurements into components of systems-level understanding, providing deeper insights into RPS6KA2 function within complex biological contexts.

How can RPS6KA2 antibodies contribute to understanding dysregulated MAPK signaling in pathological conditions?

RPS6KA2 antibodies provide valuable tools for investigating pathological MAPK signaling in various diseases:

• Cancer research applications: HRP-conjugated RPS6KA2 antibodies can help quantify expression and activation changes in tumors with MAPK pathway dysregulation. Correlating RPS6KA2 levels with patient outcomes may identify prognostic biomarkers or therapeutic targets.

• Neurodegenerative disease investigation: Given the role of MAPK signaling in neuronal function, RPS6KA2 antibodies can help characterize pathway alterations in conditions like Alzheimer's and Parkinson's diseases, potentially revealing disease-specific signaling signatures.

• Inflammatory disorder analysis: RPS6KA2 participates in signaling networks that regulate inflammatory responses. Antibody-based detection in tissues from inflammatory conditions may reveal pathway-specific dysregulation that could be therapeutically targeted.

• Longitudinal disease progression studies: Using standardized RPS6KA2 antibody protocols in longitudinal samples can track how signaling changes correlate with disease progression, potentially identifying critical transition points.

• Therapeutic response monitoring: In treatment settings, particularly with MAPK pathway inhibitors, RPS6KA2 antibodies can help monitor target engagement and pathway reactivation, informing treatment adjustments.

• Biomarker development: Validated RPS6KA2 antibodies with consistent performance characteristics could be developed into clinical diagnostic assays for conditions where MAPK pathway activity serves as a biomarker.

These applications extend RPS6KA2 antibody utility beyond basic research into translational and clinical domains, potentially contributing to improved disease understanding and therapeutic strategies.

What are the essential validation steps researchers should perform before using a new RPS6KA2 antibody in critical experiments?

Before incorporating a new RPS6KA2 antibody into important research, researchers should perform these essential validation steps:

• Specificity verification: Confirm the antibody detects the expected 83kDa band in Western blotting using positive control samples (e.g., cell lines with known RPS6KA2 expression). Verify minimal cross-reactivity with other RSK family members.

• Sensitivity assessment: Determine the limit of detection using dilution series of recombinant protein or lysates from cells with varying expression levels.

• Application-specific validation: Test the antibody in all intended applications (Western blot, IHC, IF, ELISA) following recommended dilutions (e.g., 1:500-1:2000 for WB, 1:100-1:200 for IHC) .

• Epitope accessibility confirmation: For the HRP-conjugated antibody targeting AA 228-395 , verify that this region is accessible in your experimental system and not masked by fixation or protein interactions.

• Lot-to-lot consistency testing: If transitioning between antibody lots, perform side-by-side comparisons to ensure consistent performance.

• Negative controls: Test the antibody in systems with RPS6KA2 knockdown or knockout to confirm specificity.

• Blocking peptide competition: If available, perform competition assays with the immunizing peptide to verify binding specificity.

• Cross-species reactivity verification: If working with non-human samples, confirm the antibody recognizes the orthologous protein despite potential sequence variations.

These validation steps ensure experimental reliability and facilitate accurate interpretation of results across different experimental conditions.

What is the recommended experimental workflow for comparing RPS6KA2 expression across multiple experimental conditions?

A robust workflow for comparing RPS6KA2 expression across conditions incorporates these methodological elements:

• Experimental design optimization:

  • Include biological replicates (minimum n=3) for statistical validity

  • Process all samples simultaneously to minimize technical variation

  • Include appropriate positive and negative controls

• Sample preparation standardization:

  • Use consistent lysis buffers and protein extraction protocols

  • Quantify protein concentration using the same method across all samples

  • Normalize loading amounts (25μg per lane recommended)

• Western blotting protocol:

  • Run all samples on the same gel when possible

  • Include ladder markers on each gel for accurate size determination

  • Transfer all samples simultaneously under identical conditions

• Antibody incubation optimization:

  • Use consistent antibody dilution (1:1000 recommended)

  • Apply the same incubation time and temperature across all membranes

  • Use the same lot of HRP-conjugated RPS6KA2 antibody

• Signal detection standardization:

  • Use identical exposure settings for all samples

  • Ensure detection is within the linear range (not saturated)

  • Apply consistent image analysis parameters

• Normalization approach:

  • Include loading controls (β-actin, GAPDH) for normalization

  • Apply the same quantification method across all blots

  • Consider normalization to total protein (stain-free technologies)

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Account for multiple comparisons when necessary

  • Report both statistical significance and effect size

How should researchers report RPS6KA2 antibody data in publications to ensure reproducibility?

To ensure reproducibility, researchers should include these critical details when reporting RPS6KA2 antibody data:

• Antibody specifications:

  • Complete catalog information (e.g., ABIN7168179)

  • Host species and clonality (e.g., rabbit polyclonal)

  • Epitope details (e.g., AA 228-395 or AA 415-734)

  • Conjugation information (HRP-conjugated or unconjugated)

  • Lot number and manufacturer

• Validation evidence:

  • Specificity confirmation methods

  • References to previous validation studies

  • Supporting data for new applications

• Experimental conditions:

  • Detailed sample preparation protocols

  • Exact dilutions used (e.g., 1:1000 for WB, 1:100 for IHC)

  • Incubation times and temperatures

  • Buffer compositions (e.g., 3% nonfat dry milk in TBST)

• Detection parameters:

  • Detailed description of detection system

  • Exposure times or image acquisition settings

  • Image processing methods and software

• Controls implemented:

  • Positive and negative control details

  • Loading control information

  • Technical and biological replicate numbers

• Quantification methodology:

  • Software and parameters used for densitometry

  • Normalization approach

  • Statistical analysis methods

• Limitations and constraints:

  • Known cross-reactivity issues

  • Detection limitations

  • Technical challenges encountered