RPS6KA2 Antibody, Biotin conjugated

What is RPS6KA2 and what cellular functions does it regulate?

RPS6KA2 (Ribosomal Protein S6 Kinase A2), also known as RSK3, is a member of the RSK (ribosomal S6 kinase) family of serine/threonine kinases. It functions as a signal-transducing intermediate in cellular responses to several growth factors and acts downstream of ERK (MAPK1/ERK2 and MAPK3/ERK1) signaling . This kinase contains two non-identical kinase catalytic domains and phosphorylates various substrates, including members of the mitogen-activated kinase (MAPK) signaling pathway .

RPS6KA2 plays critical roles in:

• Mediating mitogenic and stress-induced activation of transcription factors

• Regulating translation

• Mediating cellular proliferation, survival, and differentiation

• Potentially functioning as a tumor suppressor in epithelial ovarian cancer cells

The protein is found in many tissues but shows higher expression levels in lung and skeletal muscle .

What are the key specifications of commercially available RPS6KA2 antibody, biotin conjugated?

Currently available RPS6KA2 antibody, biotin conjugated products have the following specifications:

The antibody recognizes the amino acid region 228-395 of the human RPS6KA2 protein . Some versions target slightly different epitopes, such as AA 221-350 , but all maintain specificity for the RPS6KA2 protein.

What are the primary applications for RPS6KA2 antibody, biotin conjugated in research settings?

The primary applications for RPS6KA2 antibody, biotin conjugated include:

• ELISA (Enzyme-Linked Immunosorbent Assay): The biotin conjugation enables high-sensitivity detection when used with streptavidin-based detection systems .

• Immunohistochemistry (IHC): Some versions are validated for both paraffin-embedded (IHC-P) and frozen section (IHC-F) applications, allowing researchers to study RPS6KA2 localization and expression in tissue samples .

• Multi-label immunostaining: The biotin conjugation facilitates compatibility with various detection systems, making it ideal for multi-label experiments where several proteins need to be visualized simultaneously.

• Protein-protein interaction studies: When used with appropriate streptavidin-linked capture systems, these antibodies can assist in studying RPS6KA2 interactions with other signaling proteins.

The methodological approach should be optimized based on your specific research question and experimental system.

How can I optimize RPS6KA2 antibody, biotin conjugated for immunohistochemistry in different tissue types?

Optimizing RPS6KA2 antibody, biotin conjugated for immunohistochemistry requires systematic adjustment of several parameters based on tissue type:

For paraffin-embedded tissues:

• Antigen retrieval: Test both heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) to determine optimal conditions for RPS6KA2 detection.

• Antibody dilution: Begin with a dilution series (1:100, 1:200, 1:500) to establish optimal signal-to-noise ratio. The specific dilution may vary based on the antibody lot and tissue type .

• Blocking: Implement an avidin-biotin blocking step before primary antibody incubation to reduce endogenous biotin background, particularly critical in biotin-rich tissues like liver, kidney, and brain.

• Incubation time and temperature: Test both overnight incubation at 4°C and 1-2 hour incubation at room temperature to determine optimal conditions.

For frozen sections:

• Fixation: Compare 4% paraformaldehyde (10 minutes) versus acetone fixation (10 minutes) to preserve both antigenicity and tissue morphology.

• Permeabilization: Optimize Triton X-100 concentration (0.1-0.3%) to facilitate antibody penetration without compromising tissue structure.

• Signal amplification: Consider using streptavidin-conjugated fluorophores or enzymes (HRP) based on detection method requirements.

When comparing results across different tissue types, incorporate appropriate positive controls (tissues known to express RPS6KA2, particularly lung and skeletal muscle) and negative controls (antibody diluent only) to validate staining specificity.

What are the most effective methods to validate the specificity of RPS6KA2 antibody, biotin conjugated?

Validating the specificity of RPS6KA2 antibody, biotin conjugated requires a multi-faceted approach:

• Western blotting with recombinant protein:

  • Test against purified recombinant RPS6KA2 protein

  • Use recombinant proteins of closely related family members (RPS6KA1, RPS6KA3) as negative controls

  • Verify the detection of the expected ~90 kDa band for RPS6KA2

• siRNA/shRNA knockdown validation:

  • Transfect cells with RPS6KA2-specific siRNA/shRNA

  • Confirm reduction in signal compared to scrambled control

  • This approach verifies antibody specificity in cellular contexts

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (aa 228-395)

  • Compare with non-competed antibody in parallel experiments

  • Specific signal should be significantly reduced/eliminated in the competed sample

• Cross-reactivity assessment:

  • Test antibody reactivity in cells from multiple species to confirm predicted reactivity patterns

  • Verify absence of signal in species not predicted to cross-react

  • For human-specific antibodies, test in human vs. non-human cell lines

• Orthogonal method validation:

  • Compare results with alternative RPS6KA2 antibodies targeting different epitopes

  • Verify concordance of expression patterns across different detection methods (IHC, IF, WB)

When documenting validation results, include detailed experimental conditions and quantitative comparisons to establish confidence in antibody specificity.

How can I effectively troubleshoot non-specific background when using RPS6KA2 antibody, biotin conjugated in immunoassays?

Non-specific background when using biotin-conjugated RPS6KA2 antibody requires systematic troubleshooting:

Problem 1: High endogenous biotin interference

• Approach: Implement avidin-biotin blocking steps before antibody incubation

• Methodology: Incubate samples with avidin solution (15 minutes), wash, then biotin solution (15 minutes), wash again before applying antibody

• Analysis: Compare signal with and without blocking to determine effectiveness

Problem 2: Insufficient blocking of non-specific binding sites

• Approach: Optimize blocking buffer composition

• Methodology: Test different blocking agents (5% BSA, 5-10% normal serum from the same species as secondary reagent, commercial blocking buffers)

• Analysis: Compare signal-to-noise ratio across different blocking conditions

Problem 3: Cross-reactivity with similar epitopes

• Approach: Increase stringency of washing steps

• Methodology: Increase salt concentration in wash buffer (up to 500mM NaCl), add 0.1-0.3% Tween-20, increase number of washes

• Analysis: Evaluate reduction in background while monitoring specific signal retention

Problem 4: Secondary detection system issues

• Approach: Optimize streptavidin-conjugate concentration

• Methodology: Test dilution series of streptavidin-HRP or streptavidin-fluorophore

• Analysis: Determine minimal concentration that maintains specific signal while reducing background

Problem 5: Tissue-specific autofluorescence (for fluorescent detection)

• Approach: Implement autofluorescence quenching methods

• Methodology: Pre-treat sections with Sudan Black B (0.1-0.3%) or commercial autofluorescence quenchers

• Analysis: Compare signal-to-background ratio before and after treatment

Document all optimization steps systematically, including images before and after optimization to demonstrate improvement in signal specificity.

What controls should be included when designing experiments with RPS6KA2 antibody, biotin conjugated?

A robust experimental design with RPS6KA2 antibody, biotin conjugated requires several critical controls:

Essential controls for all applications:

• Primary antibody omission control:

  • Process samples identically but substitute antibody diluent for RPS6KA2 antibody

  • Evaluates background from secondary detection system alone

• Isotype control:

  • Use biotin-conjugated rabbit IgG at the same concentration

  • Detects non-specific binding due to antibody class properties rather than antigen specificity

• Positive tissue/cell control:

  • Include samples known to express RPS6KA2 (lung or skeletal muscle tissue)

  • Validates that the detection system is functioning properly

• Negative tissue/cell control:

  • Include samples with minimal RPS6KA2 expression

  • Confirms specificity of observed signals

Additional application-specific controls:

For ELISA:

• Standard curve using recombinant RPS6KA2 protein

• Serial dilution of samples to confirm linear detection range

For IHC/ICC:

• Peptide competition control (pre-incubation with immunizing peptide)

• Adjacent section stained with alternative RPS6KA2 antibody targeting different epitope

For multiplexing experiments:

• Single-stained controls for each antibody to assess bleed-through/crosstalk

• Secondary-only controls for each detection channel

What is the optimal protocol for using RPS6KA2 antibody, biotin conjugated in co-immunoprecipitation studies?

Using RPS6KA2 antibody, biotin conjugated for co-immunoprecipitation (co-IP) requires a specialized protocol that leverages the biotin-streptavidin interaction:

Reagents needed:

• RPS6KA2 antibody, biotin conjugated

• Streptavidin-coated magnetic beads

• Cell/tissue lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease and phosphatase inhibitors)

• Wash buffers of increasing stringency

Protocol:

• Sample preparation:

  • Lyse cells in ice-cold lysis buffer (1 ml per 10^7 cells)

  • Incubate 30 minutes on ice with occasional mixing

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Transfer supernatant to new tube and determine protein concentration

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with 50 μl streptavidin beads for 1 hour at 4°C

  • Remove beads by magnetic separation

  • Transfer pre-cleared lysate to new tube

• Antibody-antigen binding:

  • Add 2-5 μg biotin-conjugated RPS6KA2 antibody to 500 μg pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

• Immunoprecipitation:

  • Add 50 μl streptavidin magnetic beads

  • Incubate 2 hours at 4°C with gentle rotation

  • Collect beads using magnetic stand

• Washing:

  • Wash beads 4 times with progressively stringent wash buffers:
    a) Lysis buffer
    b) Lysis buffer with 300 mM NaCl
    c) Lysis buffer with 300 mM NaCl and 0.1% SDS
    d) 50 mM Tris-HCl, pH 7.4, 150 mM NaCl

• Elution and analysis:

  • Elute bound proteins with 50 μl 2X SDS sample buffer at 95°C for 5 minutes

  • Analyze by SDS-PAGE and Western blotting using antibodies against suspected interaction partners

Critical considerations:

• Include a non-biotin-conjugated antibody control to assess non-specific pull-down

• Incorporate a pre-immunization serum control to establish baseline

• Cross-validate findings with reverse co-IP using antibodies against suspected interaction partners

This protocol is specifically optimized to leverage the biotin conjugation of the RPS6KA2 antibody while minimizing background from non-specific interactions.

How can I differentiate between RPS6KA2 and other RSK family members in my experimental analyses?

Differentiating between RPS6KA2 (RSK3) and other RSK family members requires careful consideration of antibody specificity and experimental design:

Antibody-based differentiation:

• Epitope selection: The RPS6KA2 antibody targeting AA 228-395 or 221-350 recognizes a region with lower sequence homology to other RSK family members.

• Western blot analysis: RPS6KA2 migrates at approximately 90 kDa, but subtle size differences between family members may not be sufficient for definitive identification .

• Immunohistochemical patterns: Compare staining patterns with known tissue distribution profiles:

  • RPS6KA2/RSK3: Higher expression in lung and skeletal muscle

  • RPS6KA1/RSK1: Broadly expressed

  • RPS6KA3/RSK2: High expression in brain (mutations cause Coffin-Lowry syndrome)

  • RPS6KA6/RSK4: Restricted expression pattern

Molecular approaches for definitive differentiation:

• Isoform-specific knockdown validation:

  • Design siRNAs targeting unique regions of RPS6KA2 mRNA

  • Confirm specificity by measuring expression of all RSK family members after knockdown

  • Validate antibody specificity by demonstrating reduced signal with RPS6KA2 knockdown but not with knockdown of other RSK isoforms

• Mass spectrometry characterization:

  • After immunoprecipitation with RPS6KA2 antibody, perform tryptic digestion

  • Analyze peptide fragments by mass spectrometry

  • Identify isoform-specific peptides that uniquely distinguish RPS6KA2 from other family members

• Functional differentiation:

  • Utilize the known differences in subcellular localization between RSK family members

  • Employ cellular fractionation followed by Western blotting to assess distribution patterns

  • Correlate phosphorylation status with activation of downstream targets unique to specific RSK isoforms

When publishing results, clearly document the validation steps taken to ensure specificity for RPS6KA2 over other RSK family members.

What are the advantages and limitations of using biotin-conjugated versus unconjugated RPS6KA2 antibodies?

The choice between biotin-conjugated and unconjugated RPS6KA2 antibodies has significant implications for experimental design and outcomes:

Advantages of biotin-conjugated RPS6KA2 antibodies:

• Signal amplification: Biotin-streptavidin interaction allows for signal enhancement due to multiple streptavidin molecules binding to each biotin, improving detection sensitivity .

• Versatility in detection systems: Compatible with various streptavidin-conjugated reporter molecules (HRP, fluorophores, gold particles), providing flexibility across different detection platforms.

• Multiplexing capacity: Enables simultaneous detection of multiple targets when used with differently labeled streptavidin conjugates and primary antibodies of different species origins.

• Direct capture capability: Can be directly captured on streptavidin-coated surfaces for immunoprecipitation or immobilization applications without secondary antibodies.

• Reduced background in certain contexts: Eliminates potential cross-reactivity from species-specific secondary antibodies.

Limitations of biotin-conjugated RPS6KA2 antibodies:

• Endogenous biotin interference: Tissues with high endogenous biotin (kidney, liver, brain) may generate significant background, requiring additional blocking steps .

• Conjugation effects on binding affinity: The biotin conjugation process may alter antibody binding characteristics, potentially reducing affinity or accessing certain epitopes .

• Reduced flexibility for amplification strategies: The predetermined biotin conjugation limits options for alternative amplification methods.

• Storage stability concerns: Conjugated antibodies may have different stability profiles compared to unconjugated versions, potentially requiring more careful storage conditions .

• Incompatibility with biotin-based protein tags: Cannot be used to detect biotin-tagged proteins due to interference.

Comparative performance table:

When selecting between these options, researchers should consider their specific experimental requirements, tissue types, and detection methods.

How can I adapt RPS6KA2 antibody, biotin conjugated for use in flow cytometry applications?

Adapting biotin-conjugated RPS6KA2 antibody for flow cytometry requires specific protocol modifications to ensure optimal intracellular staining:

Protocol for intracellular RPS6KA2 detection by flow cytometry:

• Cell preparation and fixation:

  • Harvest 1×10^6 cells per sample

  • Wash cells with PBS containing 1% BSA

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash twice with PBS

• Permeabilization optimization:

  • For cytoplasmic/nuclear proteins like RPS6KA2, compare:
    a) 0.1% Triton X-100 (15 minutes)
    b) 90% ice-cold methanol (30 minutes at -20°C)
    c) Commercial permeabilization buffers

  • The optimal method depends on subcellular localization of RPS6KA2 in your specific cell type

• Blocking endogenous biotin:

  • Incubate cells with streptavidin (10 μg/ml) for 15 minutes

  • Wash twice with PBS/1% BSA

  • Incubate with free biotin (50 μg/ml) for 15 minutes

  • Wash twice with PBS/1% BSA

• Antibody staining:

  • Incubate with biotin-conjugated RPS6KA2 antibody

  • Start with 1:100 dilution in PBS/1% BSA/0.1% Triton X-100

  • Incubate 1 hour at room temperature or overnight at 4°C

  • Wash twice with PBS/1% BSA

• Detection:

  • Incubate with fluorochrome-conjugated streptavidin (e.g., streptavidin-PE or streptavidin-APC)

  • Use at manufacturer's recommended concentration (typically 1:200-1:500)

  • Incubate 30 minutes at room temperature in the dark

  • Wash twice with PBS/1% BSA

• Data acquisition optimization:

  • Include compensation controls if multiplexing

  • Use FMO (fluorescence minus one) controls to set gates

  • Include unstained, secondary-only, and isotype controls

Validation approaches:

• Confirm specificity by analyzing cells with known differential expression of RPS6KA2

• Validate results with RPS6KA2 knockdown or overexpression controls

• Compare staining patterns with unconjugated RPS6KA2 antibodies

Troubleshooting guidance:

• If signal is weak: increase antibody concentration, extend incubation time, or try alternative permeabilization methods

• If background is high: increase blocking stringency, reduce antibody concentration, or add 2% normal serum to staining buffer

This methodology is particularly useful for correlating RPS6KA2 expression with other cellular markers in heterogeneous cell populations or analyzing changes in expression following various treatments.

How does RPS6KA2 antibody, biotin conjugated compare to other methods for studying RPS6KA2 expression and function?

RPS6KA2 antibody, biotin conjugated represents one of several approaches for studying this kinase. Below is a comparative analysis with alternative methodologies:

Comparative methodological approaches for RPS6KA2 analysis:

Methodological decision framework:

• For expression analysis:

  • If protein localization is critical: Use biotin-conjugated RPS6KA2 antibody for IHC/ICC

  • If quantitative expression across samples is needed: RT-qPCR followed by Western blot validation

  • If expression in heterogeneous populations matters: Flow cytometry with biotin-conjugated antibody

• For functional analysis:

  • If activation status is primary: Use phospho-specific antibodies

  • If direct activity measurement is required: Kinase activity assays

  • If real-time monitoring is needed: Live-cell imaging with genetic reporters

• For interaction studies:

  • If detecting protein complexes: Co-IP with biotin-conjugated antibody

  • If identifying novel interactions: Mass spectrometry following antibody pull-down

  • If visualizing co-localization: Multi-label immunofluorescence with biotin-conjugated antibody

The choice of method should be guided by the specific research question, available resources, and technical expertise.

What are the most effective strategies for multiplexing RPS6KA2 antibody, biotin conjugated with other antibodies?

Multiplexing RPS6KA2 antibody, biotin conjugated with other antibodies requires strategic planning to avoid cross-reactivity and signal interference:

Optimal multiplexing strategies:

• Sequential detection approach:

  • Apply primary antibodies sequentially rather than simultaneously

  • Complete each detection cycle (primary Ab → detection system → visualization) before beginning the next

  • Between cycles, perform stringent washing or elution to remove previous antibodies

  • This approach minimizes cross-reactivity between detection systems

• Species-based multiplexing:

  • Pair biotin-conjugated RPS6KA2 antibody (rabbit host) with primary antibodies from different species (mouse, goat, etc.)

  • Use species-specific secondary antibodies for the non-biotin-conjugated primaries

  • Employ streptavidin-conjugated fluorophore with a distinct spectrum for RPS6KA2 detection

  • Example combination: Rabbit biotin-RPS6KA2 (detect with streptavidin-Cy5) + Mouse anti-ERK (detect with anti-mouse-FITC)

• Tyramide signal amplification (TSA) multiplexing:

  • Utilize sequential TSA reactions with biotin-conjugated RPS6KA2 antibody

  • After each TSA reaction, inactivate HRP with hydrogen peroxide treatment

  • This enables multiple antibodies from the same species to be used together

Protocol for immunofluorescence multiplexing:

• Sample preparation:

  • Fix and permeabilize cells/tissues using standard methods

  • Block with 5% normal serum from all secondary antibody species + avidin-biotin blocking

• Primary antibody application (Option 1 - Simultaneous):

  • Mix biotin-conjugated RPS6KA2 antibody with primary antibody of different species

  • Apply to sample and incubate overnight at 4°C

  • Wash extensively (4-5 times, 5 minutes each)

• Primary antibody application (Option 2 - Sequential):

  • Apply first primary antibody and complete detection

  • Wash extensively

  • Apply biotin-conjugated RPS6KA2 antibody

• Detection system:

  • Apply mixture of appropriate detection reagents:
    a) Streptavidin-conjugated fluorophore (e.g., streptavidin-Cy5)
    b) Species-specific secondary antibody with different fluorophore (e.g., anti-mouse-FITC)

  • Incubate 1 hour at room temperature

  • Wash extensively

• Controls for multiplexing:

  • Single-stained controls for each antibody

  • Secondary-only controls

  • Absorption controls to verify absence of cross-reactivity

Spectral considerations table:

By carefully selecting complementary detection systems and implementing appropriate controls, researchers can effectively multiplex biotin-conjugated RPS6KA2 antibody with other antibodies to examine co-expression, co-localization, and functional relationships.

How can I integrate RPS6KA2 antibody detection with phosphoproteomic analyses to understand kinase signaling networks?

Integrating RPS6KA2 antibody detection with phosphoproteomics provides a comprehensive view of kinase signaling networks:

Integrated methodological workflow:

• Parallel sample processing:

  • Split biological samples for antibody-based detection and phosphoproteomic analysis

  • Ensure identical treatment conditions to maintain data comparability

  • Process at matched time points to capture temporal dynamics

• Antibody-based RPS6KA2 profiling:

  • Use biotin-conjugated RPS6KA2 antibody for immunoprecipitation to isolate RPS6KA2 and its binding partners

  • Perform Western blotting with phospho-specific antibodies to assess RPS6KA2 activation status

  • Quantify total RPS6KA2 expression levels across experimental conditions

• Phosphoproteomic sample preparation:

  • Lyse cells in urea buffer with phosphatase inhibitors

  • Perform protein digestion (typically with trypsin)

  • Enrich for phosphopeptides using:
    a) Immobilized metal affinity chromatography (IMAC)
    b) Titanium dioxide (TiO2) enrichment
    c) Phospho-tyrosine specific antibodies for tyrosine phosphorylation

• Mass spectrometry analysis:

  • Analyze enriched phosphopeptides by LC-MS/MS

  • Identify phosphorylation sites using database search algorithms

  • Quantify phosphopeptide abundance across conditions

• Integrated data analysis:

  • Map phosphorylation changes to known RPS6KA2 substrates

  • Identify phosphorylation motifs consistent with RPS6KA2 activity (Arg-X-X-pSer/pThr)

  • Perform pathway enrichment analysis on differential phosphoproteome

  • Correlate RPS6KA2 activation status with substrate phosphorylation patterns

• Validation of RPS6KA2-specific effects:

  • Perform parallel analyses with RPS6KA2 inhibition (pharmacological or genetic)

  • Identify phosphosites specifically affected by RPS6KA2 manipulation

  • Validate key findings using targeted approaches (site-specific phospho-antibodies)

Data integration framework:

This integrated approach bridges traditional antibody-based methods with modern phosphoproteomics to provide comprehensive insights into RPS6KA2 function within signaling networks.

What are the emerging applications of RPS6KA2 antibody, biotin conjugated in cancer research and potential therapeutic development?

RPS6KA2 antibody, biotin conjugated is becoming an important tool in cancer research, with several emerging applications:

Current applications in cancer research:

• Tumor biomarker evaluation:

  • RPS6KA2 has been identified as a potential tumor suppressor in epithelial ovarian cancer

  • Biotin-conjugated antibodies facilitate high-sensitivity detection in tissue microarrays

  • Enables correlation of expression patterns with clinical outcomes across large patient cohorts

• Signaling pathway analysis:

  • RPS6KA2 functions downstream of the MAPK/ERK pathway, which is frequently dysregulated in cancer

  • Biotin-conjugated antibodies allow simultaneous detection of RPS6KA2 with other pathway components

  • Helps identify altered signaling nodes that could serve as therapeutic targets

• Drug response prediction:

  • RPS6KA2 activity may influence response to MAPK pathway inhibitors

  • Antibody-based screening can identify tumors with altered RPS6KA2 expression

  • Potential stratification marker for targeted therapy selection

Emerging methodological approaches:

• Single-cell analysis integration:

  • Combining biotin-conjugated RPS6KA2 antibody with single-cell technologies

  • Allows mapping of RPS6KA2 expression across heterogeneous tumor microenvironments

  • Protocol adaptation: Lower antibody concentrations (1:500-1:1000), shorter incubation (2-4 hours), miniaturized reaction volumes

• Extracellular vesicle (EV) detection:

  • Using biotin-conjugated antibodies to detect RPS6KA2 in tumor-derived EVs

  • Potential liquid biopsy application for non-invasive monitoring

  • Methodology: EV isolation by ultracentrifugation, permeabilization, antibody staining, analysis by high-resolution flow cytometry

• Therapeutic antibody development pipeline:

  • Biotin-conjugated research antibodies serve as starting points for therapeutic development

  • Epitope mapping using these antibodies identifies critical functional domains

  • In vitro screening with biotin-conjugated antibodies helps select candidates for humanization

Future research directions table: