RIOK2 Antibody, HRP conjugated

What is RIOK2 and why is it significant in research?

RIOK2 (RIO Kinase 2) is a serine/threonine protein kinase (EC 2.7.11.1) that has emerged as a significant research target due to its roles in multiple cellular processes. This protein has been implicated in cancer progression, particularly in glioblastoma, where it drives cell proliferation by modulating MYC and interacting with RNA-binding proteins . RIOK2's significance stems from its dual role in normal cellular functions like ribosome biogenesis and pathological processes like tumorigenesis, making it an attractive target for both basic research and potential therapeutic development .

What are the standard applications for RIOK2 antibody, HRP conjugated?

The RIOK2 antibody with HRP conjugation is primarily designed for Enzyme-Linked Immunosorbent Assay (ELISA) applications . HRP conjugation enables signal amplification when appropriate substrates are added, allowing for sensitive detection of RIOK2 protein. While the product information specifically mentions ELISA, HRP-conjugated antibodies generally are valuable tools in multiple applications including Western blotting, immunohistochemistry (IHC-P), and immunocytochemistry (ICC), providing researchers flexibility in experimental design based on their specific requirements .

How does the HRP conjugation benefit RIOK2 detection protocols?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages for RIOK2 detection:

• Signal Amplification: HRP utilizes hydrogen peroxide to oxidize various substrates (chromogenic, fluorogenic, or chemiluminescent), creating a cascade effect that significantly amplifies the detection signal, enhancing sensitivity for low-abundance RIOK2 samples .

• Stability: HRP conjugates demonstrate excellent long-term stability in storage, making them reliable for repeated use in RIOK2 detection protocols over extended research periods .

• Versatility: The conjugation enables visualization across multiple detection platforms depending on the substrate used, allowing researchers to adapt their RIOK2 detection method to available equipment and experimental needs .

• Cost-effectiveness: The regenerative nature of the HRP reaction makes this detection method economical for routine RIOK2 protein visualization in large-scale studies .

What are the proper storage and handling conditions for maintaining RIOK2 antibody activity?

To maintain optimal activity of the RIOK2 antibody with HRP conjugation, researchers should adhere to the following storage and handling protocols:

• Temperature: Upon receipt, store the antibody at -20°C or -80°C for long-term preservation .

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as these can compromise antibody integrity and reduce detection sensitivity .

• Buffer conditions: The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability .

• Working aliquots: For frequent use, consider preparing small working aliquots to minimize freeze-thaw cycles of the stock solution.

• Handling: When working with the antibody, maintain cold chain integrity and avoid contamination by using sterile technique.

How can RIOK2 antibody be used to investigate its role in cancer progression pathways?

RIOK2 antibody (HRP conjugated) can be strategically employed to elucidate RIOK2's role in cancer progression through several advanced methodological approaches:

• TORC2-Akt Signaling Investigation: Research demonstrates that RIOK2 binds to and activates the mTOR complex 2 (TORC2), which phosphorylates AKT to drive tumorigenesis. The antibody can be used in co-immunoprecipitation experiments followed by Western blot analysis to detect these interactions and assess how manipulating RIOK2 affects this signaling axis .

• MYC Modulation Studies: Given that RIOK2 influences MYC mRNA and protein levels, researchers can employ the antibody in chromatin immunoprecipitation (ChIP) assays or RNA immunoprecipitation (RIP) experiments to investigate the mechanisms through which RIOK2 regulates this critical oncogene .

• Catalytic Activity Assessment: Studies show that RIOK2's catalytic function is necessary for tumorigenesis. The antibody can help quantify wild-type versus catalytically inactive RIOK2 expression in experimental models (like the RIOK2123A,246A mutant) to correlate kinase activity with cancer progression markers .

• RNA-Binding Protein Interaction Analysis: RIOK2 interacts with RNA-binding proteins such as IMP3 in a manner dependent on its catalytic activity. The antibody can be used in proximity ligation assays to visualize and quantify these interactions in situ within tumor samples .

What methodological considerations should be addressed when using RIOK2 antibody in glioblastoma research?

When employing RIOK2 antibody in glioblastoma research, several methodological considerations require careful attention:

• PTEN Status Determination: Research indicates differential RIOK2 dependency based on PTEN mutation status. Researchers should first characterize the PTEN status of their glioblastoma models, as PTEN-null cells (like U87MG) show particular sensitivity to RIOK2 levels .

• Kinase-Dead Controls: Include catalytically inactive RIOK2 (RIOK2123A,246A) as a negative control in experiments to distinguish between kinase-dependent and independent functions, as this mutant fails to promote tumorigenesis despite protein expression .

• Apoptosis Inhibition: When investigating RIOK2's role in cellular processes other than survival, consider using caspase inhibitors like zVAD to block apoptosis, allowing for the study of non-survival functions without cell death confounding results .

• Ribosome Assembly Assessment: While RIOK2 has known functions in ribosome assembly, research suggests this may not be its primary tumorigenic mechanism. Use polysome profiling with RIOK2 knockdown combined with appropriate controls to distinguish between ribosomal and non-ribosomal functions .

• Hypotonic Lysis Conditions: When investigating RIOK2's interactions with RNA-binding proteins, use hypotonic lysis conditions to preserve cytoplasmic RNA macromolecules and associated protein complexes .

How can RIOK2 antibody be utilized to explore the relationship between RIOK2 and RNA-binding proteins?

The RIOK2 antibody can be employed through several methodological approaches to investigate the emerging relationship between RIOK2 and RNA-binding proteins:

• Co-Immunoprecipitation with Proteomic Analysis: Perform immunoprecipitation using the RIOK2 antibody under hypotonic lysis conditions to enrich for cytoplasmic RIOK2 and its binding partners. Follow with mass spectrometry to identify associated RNA-binding proteins, including IMP3, G3BP1, ATXN2L, and ILF3 .

• Comparative Analysis with Wild-Type and Catalytically Inactive RIOK2: Design experiments comparing protein interactions between wild-type RIOK2 and RIOK2123A,246A to determine which RNA-binding protein associations depend on RIOK2's catalytic activity. Research shows IMP3 associates with RIOK2 in a manner dependent on catalytic function .

• RNA-Protein Complex Identification: Combine the antibody with RNA-immunoprecipitation techniques to isolate and identify mRNAs associated with RIOK2-RBP complexes, focusing particularly on oncogenic transcripts like MYC that may be regulated through these interactions .

• Subcellular Fractionation: Utilize the antibody in fractionation experiments to determine where within the cell these RIOK2-RBP complexes form and function, as the search results indicate these interactions occur primarily in the cytoplasm .

• Sequential Co-IP Experiments: Design sequential immunoprecipitation protocols (first with RIOK2 antibody, then with antibodies against RBPs) to purify and characterize specific subcomplexes within the larger RIOK2-RBP interaction network.

What are the experimental approaches to distinguish between RIOK2's role in ribosome assembly versus its other functions in tumor cells?

Distinguishing between RIOK2's canonical role in ribosome assembly and its other functions in tumor cells requires sophisticated experimental design:

• Polysome Profiling with Partial Knockdown: Implement graded RIOK2 knockdown strategies to identify the threshold at which tumorigenic functions are compromised versus when ribosome assembly defects become apparent. Research indicates that levels of RIOK2 knockdown sufficient to arrest GBM cell growth show only minor reductions in 40S and 60S ribosome subunits relative to monosomes .

• Ribosomal vs. Non-Ribosomal Fraction Analysis: Fractionate cells to separate ribosomal and non-ribosomal components, then use the RIOK2 antibody to quantify protein distribution between these fractions under different experimental conditions.

• Rescue Experiments with Domain Mutants: Design domain-specific RIOK2 mutants that selectively disrupt either ribosomal or non-ribosomal functions, then perform rescue experiments in RIOK2-depleted cells to determine which domains are critical for which cellular processes.

• Temporal Analysis of RIOK2 Functions: Use inducible knockdown or inhibition systems to determine the kinetics of different RIOK2-dependent processes. Ribosome assembly defects and non-ribosomal functions may have different temporal profiles.

• Specific Binding Partner Manipulation: Selectively disrupt RIOK2's interaction with known ribosome assembly factors (TSR1, LTV1, NOB1, PNO1) versus novel non-ribosomal binding partners (IMP3, G3BP1, ATXN2L, ILF3) to parse their relative contributions to tumor growth .

What controls should be included when performing ELISA with RIOK2 antibody, HRP conjugated?

When conducting ELISA with RIOK2 antibody (HRP conjugated), incorporating the following controls is essential for result validation:

• Positive Control: Include samples with confirmed RIOK2 expression, such as VCaP or COLO320 cell lysates, which have been documented to express RIOK2 protein detectable with anti-RIOK2 antibodies .

• Negative Control: Incorporate samples known to have low RIOK2 expression, such as LNCaP prostate cancer cell line lysates, which serve as negative controls in RIOK2 detection experiments .

• Primary Antibody Omission Control: Include wells that receive all reagents except the primary RIOK2 antibody to assess non-specific binding of detection reagents.

• Concentration Gradient Control: Prepare a standard curve using recombinant human RIOK2 protein at defined concentrations to enable quantitative analysis.

• Blocking Peptide Control: Pre-incubate a portion of the antibody with excess immunogen peptide (recombinant Human Serine/threonine-protein kinase RIO2 protein (316-448AA)) before adding to the wells to confirm binding specificity .

• Cross-Reactivity Control: Test the antibody against related kinases (such as RIOK1 and RIOK3) to verify target specificity, particularly since RIOK family proteins share structural similarities.

How can researchers optimize immunostaining protocols with RIOK2 antibody for different sample types?

Optimizing immunostaining protocols with RIOK2 antibody requires tailored approaches for different sample types:

• Cell Lines versus Tissue Sections:

  • For cell lines: Use 4% paraformaldehyde fixation for 15-20 minutes at room temperature, followed by 0.1% Triton X-100 permeabilization.

  • For tissue sections: Consider antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0) to expose epitopes that may be masked during formalin fixation and paraffin embedding.

• Antibody Dilution Optimization:

  • Conduct a dilution series (starting with manufacturer's recommendation of 1:100 to 1:1000) to determine optimal signal-to-noise ratio.

  • For samples with low RIOK2 expression, consider signal amplification systems like tyramide signal amplification to enhance detection.

• Blocking Optimization:

  • Use 5-10% normal serum from the same species as the secondary antibody.

  • Include 0.1-0.3% Triton X-100 in blocking buffer for balanced permeabilization.

  • Consider adding 1-2% BSA to reduce non-specific binding.

• Incubation Parameters:

  • Test both overnight incubation at 4°C and shorter incubations (1-2 hours) at room temperature.

  • For tissues with high endogenous peroxidase activity, include a quenching step (0.3% H₂O₂ in methanol) before the blocking step.

• Counterstaining Considerations:

  • Choose counterstains that complement the HRP development color.

  • Optimize counterstaining time to avoid obscuring specific RIOK2 signals.

What are the potential pitfalls when studying RIOK2-mediated signaling using the HRP-conjugated antibody?

Several methodological pitfalls must be considered when studying RIOK2-mediated signaling with the HRP-conjugated antibody:

• HRP Sensitivity to Reducing Agents: HRP activity is inhibited by reducing agents commonly used in protein sample preparation (like DTT or β-mercaptoethanol). When analyzing RIOK2 signaling in complex lysates, adjust sample preparation protocols accordingly or consider using unconjugated primary antibodies for certain applications .

• Catalytic Activity versus Protein Level Distinction: The antibody detects RIOK2 protein regardless of its catalytic status. Additional experimental approaches (such as kinase assays or phosphorylation state-specific antibodies) are needed to distinguish between catalytically active and inactive RIOK2 in signaling studies .

• RNA Dependency of Interactions: RIOK2's interactions with RNA-binding proteins may be RNA-dependent. Consider including RNase treatments in control samples to determine which protein-protein interactions are direct versus RNA-mediated .

• Signal Pathway Crosstalk: RIOK2 participates in multiple signaling pathways (TORC2-Akt, MYC regulation). When studying one pathway, consider how experimental manipulations may affect parallel pathways, potentially confounding interpretation .

• Temporal Dynamics: RIOK2 signaling may have different temporal dynamics in different contexts. Design time-course experiments rather than single time-point measurements to capture the full signaling profile.

• Endogenous HRP-like Activity: Some tumor samples may have elevated levels of endogenous peroxidase-like activity, potentially generating false positive signals. Always include appropriate negative controls and consider peroxidase quenching steps in protocols.

How does RIOK2 function compare between normal cells and cancer cells?

Research reveals significant functional differences in RIOK2 between normal and cancer cells:

• Expression Level Differences:

  • Normal cells: RIOK2 is expressed at modest levels and primarily functions in ribosome biogenesis.

  • Cancer cells: RIOK2 is often overexpressed, particularly in glioblastoma and prostate cancer cell lines like VCaP and COLO320, but not in all cancer types (e.g., LNCaP prostate cancer cells show low expression) .

• Functional Role Distinctions:

  • Normal cells: RIOK2's primary role appears to be in 40S ribosomal subunit maturation.

  • Cancer cells: RIOK2 develops additional oncogenic functions including TORC2 activation, MYC regulation, and interactions with RNA-binding proteins like IMP3 .

• Catalytic Activity Requirement:

  • Normal endothelial cells (HUVEC): Though expressing ERG (and potentially RIOK2), they don't demonstrate the same dependency on RIOK2 catalytic activity as cancer cells .

  • Cancer cells: Catalytic activity of RIOK2 is essential for driving proliferation, as evidenced by the growth-inhibitory effect of catalytically dead RIOK2123A,246A in U87MG glioblastoma cells .

• Survival Dependency:

  • Normal cells: Generally not dependent on RIOK2 for survival.

  • Cancer cells: Often show "addiction" to RIOK2 function, particularly in PTEN-null contexts, where RIOK2 knockdown or catalytic inhibition triggers growth arrest and apoptosis .

• Binding Partner Profile:

  • Normal cells: RIOK2 primarily associates with ribosome assembly factors.

  • Cancer cells: RIOK2 forms additional complexes with RNA-binding proteins involved in mRNA stability, transport, and translation .

What are the current methods to evaluate RIOK2 inhibition efficacy in experimental models?

Researchers can employ multiple methodological approaches to evaluate RIOK2 inhibition efficacy:

• Cell Viability and Proliferation Assays:

  • WST-1 assays to measure metabolic activity as a proxy for cell viability following RIOK2 inhibition.

  • Growth curve analysis comparing RIOK2 wild-type overexpression, RIOK2 knockdown, and catalytically dead RIOK2123A,246A expression .

  • IC50 determination using dose-response curves of potential RIOK2 inhibitors, as demonstrated with ERGi-USU compound in VCaP cells (which appears to act via RIOK2 inhibition with a Kd of 200 nmol/L) .

• Pathway Analysis:

  • Western blotting to assess TORC2-Akt signaling pathway components following RIOK2 inhibition.

  • qPCR and protein analysis of MYC levels, which are modulated by RIOK2 activity .

  • Analysis of downstream RNA-binding protein functions through RNA immunoprecipitation followed by sequencing.

• Biochemical Assessments:

  • Tryptophan fluorescence quenching assays to determine direct binding of inhibitors to RIOK2, as demonstrated with ERGi-USU binding to human RIOK2 but not ancestral Riok2 .

  • In vitro kinase assays to directly measure RIOK2 catalytic activity inhibition.

• Genetic Approaches:

  • Comparison of phenotypes between RIOK2 knockdown and expression of dominant-negative RIOK2123A,246A.

  • Combined knockdown experiments targeting RIOK2 along with related kinases (RIOK1, RIOK3) to assess potential compensatory mechanisms .

• Animal Model Evaluation:

  • Tumor xenograft growth assessment following RIOK2 inhibition.

  • Combination therapy approaches in animal models to determine synergistic potential with standard-of-care treatments.

How can researchers distinguish between direct and indirect effects of RIOK2 inhibition in cellular systems?

Distinguishing between direct and indirect effects of RIOK2 inhibition requires meticulous experimental design:

• Temporal Analysis:

  • Perform time-course experiments following RIOK2 inhibition to establish the sequence of cellular events.

  • Direct effects typically manifest rapidly (minutes to hours) whereas indirect effects emerge later (hours to days).

  • Use inducible expression systems for catalytically inactive RIOK2123A,246A to create precise temporal control of inhibition .

• Dose-Dependency Assessment:

  • Implement graded levels of RIOK2 inhibition through titrated RNAi or small molecule inhibitor concentrations.

  • Direct effects typically show more proportional relationships to inhibition levels than downstream indirect effects.

• Rescue Experiments:

  • Attempt to rescue phenotypes by expressing inhibitor-resistant RIOK2 mutants.

  • Direct effects should be specifically rescued by wild-type RIOK2 but not by catalytically inactive RIOK2123A,246A .

• Substrate and Binding Partner Analysis:

  • Identify direct RIOK2 substrates through phosphoproteomic analysis following acute RIOK2 inhibition.

  • Use co-immunoprecipitation with mass spectrometry to characterize how the RIOK2 interactome changes upon inhibition, focusing on both ribosomal assembly factors (TSR1, LTV1, NOB1, PNO1) and RNA-binding proteins (IMP3, G3BP1, ATXN2L, ILF3) .

• Combinatorial Approaches:

  • Combine RIOK2 inhibition with inhibitors of suspected downstream pathways.

  • If inhibiting a downstream pathway prevents certain consequences of RIOK2 inhibition, those consequences likely represent indirect effects.

How can RIOK2 antibody be used to investigate its potential as a therapeutic target in cancer?

The RIOK2 antibody can be instrumental in establishing RIOK2's potential as a therapeutic target through several methodological approaches:

• Tumor Dependency Profiling:

  • Use the antibody to catalog RIOK2 expression across diverse tumor types and correlate with clinical outcomes.

  • Develop immunohistochemical scoring systems to stratify tumors based on RIOK2 expression levels.

  • Compare RIOK2 expression in matched normal/tumor samples to establish cancer-specific overexpression patterns.

• Target Validation Studies:

  • Employ the antibody to confirm knockdown efficiency in RNAi-based synthetic lethality screens across cancer cell panels.

  • Use in immunoblotting to validate the efficacy of potential RIOK2 inhibitors like ERGi-USU, which binds RIOK2 with high affinity (Kd = 200 nmol/L) .

  • Perform immunoprecipitation studies to identify cancer-specific binding partners that might offer more selective therapeutic targeting.

• Mechanism of Action Studies:

  • Use the antibody to track RIOK2 subcellular localization changes following drug treatments.

  • Monitor RIOK2 post-translational modifications that might be induced by therapeutic agents.

  • Analyze RIOK2-containing protein complexes before and after drug treatment to understand mechanism-based resistance.

• Companion Diagnostic Development:

  • Develop standardized immunohistochemistry protocols using the antibody to identify patients likely to benefit from RIOK2-targeted therapies.

  • Create assays to monitor therapy response based on changes in RIOK2 expression or activity.

• Combination Therapy Evaluation:

  • Analyze changes in RIOK2 expression or activity following treatment with standard therapies to identify potential synergistic combinations.

  • Investigate how RIOK2 inhibition affects cellular sensitivity to other targeted agents or conventional chemotherapies.

What new methodologies are emerging for studying RIOK2's interaction with RNA-binding proteins?

Emerging methodologies for investigating RIOK2's interactions with RNA-binding proteins include:

• Proximity-Based Labeling Techniques:

  • BioID or TurboID fusion constructs with RIOK2 to identify proximal proteins in living cells.

  • APEX2-based proximity labeling to capture transient interactions between RIOK2 and RNA-binding proteins in different subcellular compartments.

  • These approaches can overcome limitations of traditional co-immunoprecipitation by capturing interactions in their native cellular context.

• Advanced RNA-Protein Interaction Mapping:

  • CLIP-seq (Cross-Linking Immunoprecipitation followed by sequencing) using RIOK2 antibodies to identify RNAs directly or indirectly associated with RIOK2 complexes.

  • PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced CLIP) to map precise RNA binding sites within RIOK2-RBP complexes.

  • RNA-protein interaction detection using MS2 tagging systems to visualize dynamic complexes in living cells.

• Single-Cell Analysis Approaches:

  • Single-cell proteomics to characterize heterogeneity in RIOK2-RBP complexes across tumor cell populations.

  • Spatial transcriptomics combined with protein analysis to map the subcellular locations of RIOK2-RBP-RNA interactions.

  • Live-cell imaging with split fluorescent protein complementation to visualize RIOK2-RBP interactions in real-time.

• Structural Biology Techniques:

  • Cryo-EM to determine the structure of RIOK2 in complex with its RNA-binding protein partners.

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces between RIOK2 and RBPs like IMP3.

  • Integrative structural modeling combining multiple data types to generate comprehensive structural models of RIOK2-RBP complexes.

• Functional Genomics Screening:

  • CRISPR interference or activation screens targeting RNA-binding proteins to systematically map functional interactions with RIOK2.

  • Domain-focused CRISPR scanning to identify specific regions required for RIOK2-RBP interactions.