RPS6KB2 Antibody Pair

What is RPS6KB2 and why is it an important research target?

RPS6KB2 (Ribosomal Protein S6 Kinase, 70kDa, Polypeptide 2) is a member of the RSK (ribosomal S6 kinase) family of serine/threonine kinases. It contains a kinase catalytic domain and phosphorylates the S6 ribosomal protein and eukaryotic translation initiation factor 4B (eIF4B) . This phosphorylation is significant because it leads to increased protein synthesis and cell proliferation . RPS6KB2 is involved in multiple critical cellular pathways including regulation of cell growth, insulin signaling, and inflammation . Recent studies have shown genetic variation in RPS6KB2 is associated with risk of developing colon cancer, making it an important target for oncology research . Additionally, it appears to act downstream of mTOR signaling in response to growth factors and nutrients to promote cell proliferation, cell growth, and cell cycle progression in an alternative pathway regulated by MEAK7 .

What distinguishes an RPS6KB2 antibody pair from single antibodies?

An RPS6KB2 antibody pair consists of two matched antibodies specifically designed to work together for optimal detection and quantification of the RPS6KB2 protein. Unlike single antibodies, these pairs typically include:

• A capture antibody (usually mouse monoclonal anti-RPS6KB2, approximately 100 μg) that binds to the target protein

• A detection antibody (typically rabbit purified polyclonal anti-RPS6KB2, approximately 50 μg) that recognizes a different epitope on the captured protein

The dual-antibody approach enhances specificity and sensitivity compared to single-antibody detection methods, particularly in sandwich ELISA applications. This paired system minimizes cross-reactivity issues and improves signal-to-noise ratios in quantitative protein analysis .

How should I design validation experiments for a new RPS6KB2 antibody pair?

When validating a new RPS6KB2 antibody pair, a systematic approach is essential:

• Positive and negative controls: Include cell lines known to express RPS6KB2 (e.g., HeLa, SH-SY5Y, HL-60, SW620, MCF-7) as positive controls . For negative controls, use either cell lines with low/no expression or perform siRNA knockdown of RPS6KB2.

• Cross-reactivity assessment: Test the antibody against samples from different species if working with non-human models. Known cross-reactivity includes human, mouse, and rat models for many RPS6KB2 antibodies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (if available) to confirm specificity.

• Multiple techniques validation: Confirm results across different methods (WB, IF, IHC) to strengthen confidence in antibody specificity.

• Molecular weight verification: Confirm detected bands match the expected molecular weight (calculated: 16 kDa/53 kDa; observed: often ~63 kDa due to post-translational modifications) .

• Phospho-specific validation: For phospho-specific antibodies, treat samples with phosphatases to demonstrate specificity for the phosphorylated form .

The validation should be documented with appropriate controls for each experimental technique planned for future use.

What are the optimal storage conditions for maintaining RPS6KB2 antibody pair activity?

Proper storage is critical for maintaining the activity and specificity of RPS6KB2 antibody pairs:

• Temperature: Store antibody reagents at -20°C or lower for long-term storage .

• Aliquoting: Divide the antibody solution into small aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade antibody performance .

• Short-term storage: For frequent use within a month, store at 4°C .

• Freeze-thaw cycles: Minimize freeze-thaw cycles; each cycle can decrease antibody activity .

• Buffer conditions: Most RPS6KB2 antibodies are supplied in phosphate buffered solution (pH 7.4) containing stabilizers (often 0.05%) and glycerol (typically 50%) .

• Shipping considerations: Upon receipt of ice-packed antibodies, immediately transfer to recommended storage temperature .

• Shelf life: Most products are valid for 12 months when stored as recommended .

Following these guidelines ensures optimal antibody performance throughout your experimental timeline.

How do I troubleshoot discrepancies between expected and observed molecular weights for RPS6KB2?

Molecular weight discrepancies are common with RPS6KB2 antibodies and require systematic troubleshooting:

• Post-translational modifications: RPS6KB2 undergoes extensive phosphorylation, which can significantly alter migration patterns. The calculated MW is often cited as 16 kDa/53 kDa, but the observed MW is frequently ~63 kDa .

• Isoform detection: Multiple isoforms (P70-beta-1, P70-beta-2) may be detected, contributing to multiple bands .

• Sample preparation effects: Different lysis buffers and denaturing conditions can affect protein migration.

• Verification strategies:

  • Use phosphatase treatment to confirm phosphorylation-dependent mobility shifts

  • Compare with recombinant proteins of known molecular weight

  • Validate with genetic approaches (overexpression or knockdown)

  • Test multiple antibodies recognizing different epitopes

• Expected variations: As noted in the Elabscience antibody documentation, "The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

These variations should be documented and considered when interpreting experimental results.

How can RPS6KB2 antibody pairs be used to study mTOR signaling pathway dynamics?

RPS6KB2 is a key downstream effector in the mTOR signaling pathway, making antibody pairs valuable tools for studying this network:

• Multiplex phosphorylation analysis: Use phospho-specific antibodies (e.g., phospho-S371 ) in combination with total RPS6KB2 antibodies to determine the activation state of the pathway. This approach allows calculation of phosphorylation-to-total protein ratios.

• Temporal dynamics: Apply time-course experiments with stimuli such as growth factors, nutrients, or insulin to track RPS6KB2 activation kinetics.

• Pharmacological intervention: Combine with mTOR inhibitors (rapamycin, torin) to assess pathway dependencies:

  • Pretreat cells with inhibitors before stimulation

  • Monitor changes in both RPS6KB2 and its substrates (S6, eIF4B)

  • Correlate with cellular outcomes like protein synthesis or proliferation

• Pathway crosstalk analysis: Simultaneously probe multiple pathway components to understand network interactions:

  • MEAK7-dependent alternative pathways

  • Integration with PI3K/Akt signaling

  • Connections to insulin response elements

• Cellular localization studies: Use immunofluorescence to track RPS6KB2 translocation between cytoplasm and nucleus under different conditions, as RPS6KB2 has been reported in both cellular compartments .

This systematic approach enables researchers to map the functional relationships within complex signaling networks.

What are the best approaches for studying RPS6KB2 phosphorylation sites using antibody techniques?

Studying RPS6KB2 phosphorylation requires specialized approaches:

• Site-specific phospho-antibodies: Several key phosphorylation sites can be monitored with specific antibodies:

  • Phospho-S371 antibodies for activation loop phosphorylation

  • Phospho-S423 antibodies for functional assessment

  • Phospho-Ser-247 antibodies for CK1-dependent regulation

• Multiplexed detection strategies:

  • Sequential reprobing of membranes with different phospho-specific antibodies

  • Multiplex flow cytometry with differently conjugated phospho-antibodies

  • Phospho-proteomic analysis combined with antibody validation

• Manipulation of phosphorylation states:

  • Phosphatase treatment controls to confirm specificity

  • Kinase inhibitor panels to identify regulatory enzymes

  • Site-directed mutagenesis (e.g., Ser→Ala mutations) to validate antibody specificity

• Integration with mass spectrometry:

  • Immunoprecipitate RPS6KB2 using antibody pairs

  • Analyze by LC-MS/MS to identify novel phosphorylation sites

  • Validate findings with phospho-specific antibodies

As shown in the research by Hutchinson et al., "We introduced individual Ser → Ala mutations at codons 235, 236, 240, 244, and 247 of rpS6 and expressed the mutant proteins in HEK 293T cells. The Ser-247 → Ala mutation ablated immunoreactivity, suggesting that the α-pPER2 antibody recognizes phosphorylated Ser-247" . This demonstrates the value of combining mutagenesis with antibody techniques for rigorous phosphorylation site analysis.

How can RPS6KB2 antibody pairs be implemented in cancer research studies?

RPS6KB2 has significant implications in cancer research, and antibody pairs can be employed in several sophisticated approaches:

• Cancer tissue profiling:

  • Use immunohistochemistry to assess RPS6KB2 expression and activation across tumor types

  • Compare with normal adjacent tissue to establish baseline expression

  • Correlate with clinical outcomes and treatment responses

• Genetic variation correlation:

  • Combine with genotyping studies to investigate associations between RPS6KB2 genetic variants and protein expression/activation

  • Recent findings indicate "genetic variation in RPS6KB2 is associated with risk of developing colon cancer"

  • Assess potential as a biomarker for cancer susceptibility or progression

• Signaling network analysis:

  • Study interactions with other cancer-related pathways

  • Investigate "significant interactions with other genes operating in similar mechanisms, including Akt1, FRAP1, NFκB1, and PIK3CA"

  • Map differential activation in treatment-resistant versus sensitive tumors

• Molecular subtyping:

  • Correlate RPS6KB2 expression/activation with specific tumor markers

  • Evidence suggests these "genes and this pathway may importantly contribute to CIMP+ tumors and tumors with KRAS2 mutations"

• Therapeutic target validation:

  • Monitor RPS6KB2 inhibition in response to targeted therapies

  • Assess combination treatments targeting multiple pathway components

  • Identify resistance mechanisms through pathway reactivation

This multifaceted approach can provide insights into both fundamental cancer biology and potential therapeutic strategies.

What are the key considerations for using RPS6KB2 antibody pairs in sandwich ELISA development?

Developing a reliable sandwich ELISA for RPS6KB2 quantification requires attention to several critical factors:

• Antibody pair selection: Choose a validated pair with demonstrated specificity, as commercial RPS6KB2 antibody pairs are specifically optimized for this application .

• Optimal working dilutions: As noted in technical documentation, "Optimal working dilution should be determined by the investigator" . Establish this through titration experiments:

  • Test various concentrations of capture and detection antibodies in a matrix format

  • Identify combinations providing maximum signal-to-noise ratio

  • Document optimal ratios in standardized protocols

• Sample preparation optimization:

  • Evaluate different cell/tissue lysis buffers for maximum protein extraction

  • Test various blocking agents to minimize background

  • Determine if sample dilution is needed to prevent hook effects

• Standard curve development:

  • Use recombinant RPS6KB2 to establish a reference curve

  • Ensure linearity across the expected concentration range

  • Include internal controls for inter-assay normalization

• Validation parameters to assess:

  • Sensitivity (lower limit of detection)

  • Specificity (cross-reactivity with related proteins)

  • Precision (intra- and inter-assay coefficients of variation)

  • Recovery (spike-in experiments with known quantities)

  • Parallelism (dilutional linearity of biological samples)

• Specific technical recommendations:

  • The reagents are typically "sufficient for at least 3-5 x 96 well plates using recommended protocols"

  • Store reagents at -20°C or lower and aliquot to avoid freeze-thaw cycles

Following these methodological considerations will help establish a robust ELISA system for RPS6KB2 quantification.

How do I select the most appropriate antibody pair for specific experimental objectives?

Selecting the optimal RPS6KB2 antibody pair requires matching technical specifications to your specific research goals:

• Application-specific considerations:

  Application	Key Selection Criteria	Examples
  ELISA	Validated matched pairs with demonstrated sensitivity	ABIN1342162 , H00006199-AP51
  WB/IP Combination	Compatibility with IP and WB buffers	H00006199-PW1
  Cellular Localization	Validated in IF/ICC applications	ABIN7270034 , E-AB-62204
  Tissue Staining	IHC-validated antibodies	CSB-PA02905A0Rb

• Target region and form:

  • Phospho-specific antibodies for activation studies (e.g., phospho-S371 , phospho-S423 )

  • Total protein antibodies for expression level studies

  • Domain-specific antibodies depending on research focus (e.g., N-terminal vs. catalytic domain)

• Species compatibility:

  • Human-only studies: Most RPS6KB2 antibodies

  • Multi-species studies: Antibodies validated in human, mouse, and rat

• Clonality considerations:

  • Monoclonal/polyclonal combinations for ELISA (increased specificity)

  • Monoclonal for consistent lot-to-lot reproducibility

  • Polyclonal for increased sensitivity in some applications

• Validation data assessment:

  • Review published literature citing specific antibody pairs

  • Examine manufacturer validation data in relevant applications

  • Consider antibodies with validation across multiple cell lines (e.g., "Verified Samples in WB: HeLa, SH-SY5Y, HL-60, SW620, MCF-7, Mouse kidney, Rat liver" )

Selecting antibodies based on these criteria will help ensure experimental success and data reliability.

What troubleshooting approaches should be used when RPS6KB2 antibody pairs show weak or inconsistent signals?

When encountering weak or inconsistent signals with RPS6KB2 antibody pairs, a systematic troubleshooting approach should be employed:

• Sample preparation issues:

  • Verify protein extraction efficiency with different lysis buffers

  • Check protein integrity using total protein stains

  • Ensure samples contain adequate RPS6KB2 levels (use positive control cell lines like HeLa or MCF-7 )

  • Consider protease/phosphatase inhibitors to preserve protein modifications

• Antibody-specific factors:

  • Verify antibody integrity (avoid repeated freeze-thaw cycles )

  • Test different antibody dilutions beyond recommended ranges

  • Consider lot-to-lot variations (request certificate of analysis from manufacturer)

  • Evaluate alternative antibody pairs targeting different epitopes

• Technical optimization:

  • For ELISA: Modify coating buffers, incubation times, washing stringency

  • For WB: Adjust transfer conditions, blocking reagents, incubation temperatures

  • For IHC/IF: Optimize antigen retrieval methods, fixation protocols

  • Test different detection systems (HRP vs. fluorescent, amplification methods)

• Biological variables:

  • Consider cell/tissue-specific RPS6KB2 expression levels

  • Evaluate post-translational modifications affecting epitope accessibility

  • Account for activation state (e.g., treatment with growth factors)

  • Check for genetic variations affecting antibody binding

• Special considerations for RPS6KB2:

  • Expected molecular weight variations (calculated: 16-53 kDa; observed: often ~63 kDa)

  • Subcellular localization effects (RPS6KB2 can be found in both nucleus and cytoplasm)

  • Phosphorylation state affecting antibody recognition

Documenting these troubleshooting steps and outcomes will help establish robust protocols for future experiments.

How are RPS6KB2 antibody pairs being used to investigate its role in non-cancer diseases?

Beyond cancer, RPS6KB2 antibody pairs are enabling investigation of this kinase in various disease contexts:

• Metabolic disorders:

  • RPS6KB2 is involved in insulin signaling and glucose metabolism

  • Antibody pairs are being used to study pathway dysregulation in diabetes models

  • Correlations between RPS6KB2 activation and insulin resistance can be quantified

• Neurodegenerative diseases:

  • Growing evidence links mTOR pathway dysregulation to neurodegeneration

  • RPS6KB2 antibodies enable assessment of protein synthesis regulation in models of Alzheimer's and Parkinson's diseases

  • Phospho-specific antibodies help track altered signaling in patient-derived samples

• Inflammatory conditions:

  • RPS6KB2 has been implicated in inflammatory pathways

  • Antibody pairs facilitate studies of activation patterns in immune cells

  • Monitoring relationships between cytokine signaling and RPS6KB2 activation

• Aging research:

  • mTOR/S6K pathways are critical regulators of cellular senescence

  • RPS6KB2 antibodies support investigation of its specific role in aging mechanisms

  • Comparative studies between RPS6KB1 (S6K1) and RPS6KB2 (S6K2) functions

• Cardiovascular diseases:

  • Emerging role in cardiac hypertrophy and remodeling

  • Antibody-based studies of expression/activation in different cardiac cell types

  • Investigation of responses to mechanical stress and hypoxia

These applications demonstrate how RPS6KB2 antibody pairs are contributing to our understanding of diverse pathological processes beyond their established role in cancer research.

What advanced techniques are being developed to enhance RPS6KB2 detection specificity and sensitivity?

Research is advancing several innovative approaches to improve RPS6KB2 detection:

• Proximity ligation assays (PLA):

  • Combining RPS6KB2 antibodies with antibodies against interaction partners

  • Enabling visualization of protein complexes in situ

  • Providing subcellular resolution of protein interactions

• Single-cell analysis technologies:

  • Adaptation of RPS6KB2 antibodies for mass cytometry (CyTOF)

  • Integration with single-cell western blotting platforms

  • Development of antibody panels for multiparameter flow cytometry

• Nanobody and single-domain antibody approaches:

  • Smaller antibody derivatives with improved tissue penetration

  • Enhanced access to conformational epitopes

  • Reduced background in imaging applications

• CRISPR-based tagging strategies:

  • Endogenous tagging of RPS6KB2

  • Validation of antibody specificity using gene-edited controls

  • Combining genetic and immunological approaches for enhanced specificity

• Microfluidic antibody-based assays:

  • Miniaturized platforms requiring minimal sample volumes

  • Integrated systems for automated sample processing

  • Higher throughput screening of RPS6KB2 modulation

• Computational antibody engineering:

  • In silico design of RPS6KB2-specific antibodies

  • Structure-based optimization of binding properties

  • Prediction of cross-reactivity with related kinases for improved specificity

These emerging technologies promise to expand both the sensitivity and specificity of RPS6KB2 detection in complex biological samples.

How can researcher-generated validation data for RPS6KB2 antibodies be effectively shared with the scientific community?

Improving RPS6KB2 antibody validation data sharing requires systematic approaches:

• Comprehensive documentation standards:

  • Detailed experimental protocols including all buffer compositions

  • Raw data sharing (unprocessed blot images, flow cytometry plots)

  • Inclusion of positive and negative controls

  • Complete antibody information (catalog number, lot number, dilution)

• Specialized antibody validation repositories:

  • Contribute to databases like Antibodypedia or the Antibody Registry

  • Upload validation data to community resources like CiteAb or Biocompare

  • Participate in antibody validation initiatives like the Antibody Validation Database

• Publication practices:

  • Include detailed antibody validation data in supplements

  • Publish dedicated antibody characterization papers for extensively validated reagents

  • Cite both successful and unsuccessful antibody applications

• Open science approaches:

  • Share protocols on platforms like protocols.io

  • Deposit raw data in appropriate repositories

  • Utilize electronic lab notebooks with sharing capabilities

• Cross-laboratory validation networks:

  • Participate in multi-lab antibody testing initiatives

  • Establish standard reference samples for comparative analysis

  • Develop consensus reporting standards for antibody performance

• Integration with -omics datasets:

  • Correlate antibody-based findings with RNA-seq or proteomics data

  • Validate specificity against knockout/knockdown datasets

  • Connect antibody validation to functional genomics resources