WDR59 Antibody, Biotin conjugated

What is WDR59 and what cellular functions does it mediate?

WDR59 (WD Repeat Domain 59) is a component of the GATOR2 complex involved in regulating TORC1 (Target of Rapamycin Complex 1) signaling. Intriguingly, WDR59 can function in both inhibitory and promotional capacities regarding TORC1 activity, depending on cellular context. In Drosophila ovaries and eye imaginal disc brain complexes, WDR59 inhibits TORC1 activity by counteracting GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body and mammalian HeLa cells, WDR59 promotes TORC1 activation by preventing proteolytic destruction of other GATOR2 components such as Mio and Wdr24 . WDR59 is localized to the lysosomal membrane and plays a crucial role in cellular response to amino acid availability .

What epitopes do commercially available biotin-conjugated WDR59 antibodies recognize?

Commercial biotin-conjugated WDR59 antibodies primarily target the amino acid region 619-862 of human WDR59 protein. These rabbit polyclonal antibodies are generated using recombinant human GATOR complex protein WDR59 as the immunogen . Other non-conjugated variants may target different regions, such as the mouse monoclonal antibody that recognizes an epitope containing the sequence "RKQKEGSKDYQLVTWSRDQTLRMWRVDSQMQRLCANDILDGVDEFIESIS" .

How does biotin conjugation affect antibody functionality in WDR59 detection?

Biotin conjugation provides significant advantages for WDR59 detection by enabling signal amplification through high-affinity binding with streptavidin-conjugated detection reagents. This modification maintains the antibody's specificity while enhancing sensitivity in detection systems. The biotin-conjugated WDR59 antibodies are protein G purified with >95% purity, preserving binding specificity to the amino acid region 619-862 . The conjugation does not interfere with epitope recognition but facilitates more flexible detection methods, particularly beneficial in ELISA applications where signal enhancement is desirable.

What are the validated applications for biotin-conjugated WDR59 antibodies?

Biotin-conjugated WDR59 antibodies are primarily validated for ELISA applications according to manufacturer specifications . This contrasts with non-biotinylated variants of WDR59 antibodies that may have broader application ranges including Western blotting (WB) and immunofluorescence (IF). For researchers requiring broader application compatibility, it's worth noting that alternative formats such as FITC-conjugated and HRP-conjugated WDR59 antibodies are also available . When designing experiments, researchers should carefully select the antibody format that best aligns with their specific application requirements.

What is the recommended protocol for optimizing ELISA with biotin-conjugated WDR59 antibodies?

For optimal ELISA performance with biotin-conjugated WDR59 antibodies, researchers should implement a systematic dilution optimization approach. Beginning with manufacturer-suggested dilutions, perform a checkerboard titration to determine the optimal antibody concentration that maximizes specific signal while minimizing background. The antibodies are supplied in a liquid format containing 50% glycerol in 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . For ELISA applications:

• Coat plates with target protein or sample

• Block with appropriate blocking buffer

• Apply biotin-conjugated WDR59 antibody at several dilutions

• Detect with streptavidin-HRP or other biotin-binding detection systems

• Develop with appropriate substrate

Optimal working dilutions should be determined empirically for each specific experimental system .

How can researchers validate antibody specificity for WDR59 in experimental systems?

To validate WDR59 antibody specificity, researchers should implement multiple complementary approaches:

• Positive and negative controls: Include known WDR59-expressing samples alongside negative controls such as WDR59 knockout or knockdown samples

• Cross-reactivity assessment: The biotin-conjugated antibodies are specifically reactive with human WDR59 , but cross-reactivity should be verified when using in various systems

• Blocking peptide competition: Using the immunogen peptide (amino acids 619-862) to compete with antibody binding

• Validation across multiple techniques: Where possible, confirm findings using alternative detection methods or antibodies targeting different epitopes of WDR59

• Molecular weight verification: When using in western blot applications, verify detection at the expected molecular weight

This multi-faceted validation approach ensures experimental rigor and reproducibility when studying WDR59.

How can WDR59 antibodies be used to study the differential effects of WDR59 on TORC1 regulation across tissue types?

To investigate the tissue-specific dual roles of WDR59 in TORC1 regulation, researchers can design comparative immunohistochemistry studies using WDR59 antibodies alongside TORC1 activity markers. Since WDR59 has been shown to promote TORC1 activity in some tissues (fat body, HeLa cells) while inhibiting it in others (ovary, eye imaginal disc) , experimental approaches should include:

• Comparative tissue analysis: Apply WDR59 detection across multiple tissue types simultaneously

• Co-localization studies: Combine WDR59 antibody staining with markers for GATOR1 and GATOR2 complex components

• Activity correlation: Correlate WDR59 levels with downstream TORC1 activity markers like phosphorylated S6K or 4E-BP1

• Genetic complementation: In tissues showing inhibitory functions, test rescue experiments using GATOR2 complex components

This approach enables mechanistic understanding of how the same protein achieves opposing regulatory functions in different cellular contexts.

What insights can be gained about the ring domain interactions of WDR59 using specialized immunoprecipitation techniques?

The Ring domains of WDR59, along with those of Mios and WDR24, are essential for amino acid-mediated mTORC1 activation . To study these interactions, researchers can employ:

• Co-immunoprecipitation with domain specificity: Using biotin-conjugated WDR59 antibodies for pull-down assays to analyze interactions between Ring domains of WDR59, WDR24, and Mios

• Proximity ligation assays: To visualize Ring domain interactions in situ

• NanoBit-based interaction assays: As demonstrated in research, to monitor Ring-Ring domain interactions in cells with high sensitivity

• Mutation analysis: Introducing mutations in conserved residues of Ring domains to assess impact on complex formation

These specialized techniques can reveal how WDR59 Ring domains facilitate the assembly and function of the GATOR2 complex, providing mechanistic insights into amino acid-sensing pathways.

How does WDR59 contribute to the regulation of GATOR1-GATOR2 complex interaction during amino acid starvation?

WDR59 plays a sophisticated role in regulating the interaction between GATOR1 and GATOR2 complexes in response to nutrient availability. Research has shown that WDR59 attenuates the binding of GATOR2 to GATOR1, which increases GATOR1's ability to bind and inhibit the Rag GTPase complex, ultimately affecting TORC1 activity . To investigate this regulatory mechanism:

• Nutrient response assays: Compare protein-protein interactions under fed versus starved conditions

• Quantitative co-immunoprecipitation: Measure GATOR1-GATOR2 association strength with and without WDR59

• Lysosomal localization studies: Track WDR59 recruitment to lysosomes under varying nutrient conditions

• Functional reconstitution: Assess the ability of wildtype versus mutant WDR59 to restore normal GATOR complex interactions

These approaches can elucidate how WDR59 functions as a molecular switch in the nutrient-sensing machinery controlling TORC1 activity.

What strategies can address non-specific binding issues with biotin-conjugated WDR59 antibodies?

When encountering non-specific binding with biotin-conjugated WDR59 antibodies, implement the following troubleshooting strategies:

• Optimize blocking conditions: Use alternative blocking agents beyond standard BSA or milk, such as casein or commercial blocking buffers

• Adjust antibody concentration: Titrate to determine the minimal effective concentration that maintains specific signal while reducing background

• Pre-adsorption: Pre-incubate antibody with tissues/cells lacking WDR59 to remove cross-reactive antibodies

• Buffer optimization: Modify salt concentration and detergent levels in wash buffers to reduce non-specific interactions

• Endogenous biotin blocking: When working with biotin-rich samples, use streptavidin/avidin pre-blocking steps to minimize background

These approaches can significantly improve signal-to-noise ratio in experiments using biotin-conjugated WDR59 antibodies.

How should researchers address variability in WDR59 detection across different cell types?

Variability in WDR59 detection across cell types may reflect biological differences in expression levels or technical challenges. To address this variability:

• Standardize protein extraction: Use protocols optimized for membrane proteins, as WDR59 localizes to lysosomal membranes

• Validate antibody performance: Test antibody performance in each cell type with appropriate positive and negative controls

• Consider fixation effects: When performing immunohistochemistry, test multiple fixation methods as membrane protein epitopes may be differentially accessible

• Normalize to loading controls: Use appropriate housekeeping proteins for quantitative comparisons

• Context-specific optimization: Remember that WDR59 functions differently across tissues , so detection protocols may need tissue-specific adjustments

This systematic approach helps distinguish between technical variability and true biological differences in WDR59 expression or localization.

How might biotin-conjugated WDR59 antibodies contribute to understanding the therapeutic targeting of mTOR signaling pathways?

Biotin-conjugated WDR59 antibodies offer unique opportunities for investigating targeted therapeutic approaches to mTOR signaling pathways:

• Drug-target interaction studies: Using these antibodies to assess how potential therapeutics affect WDR59 interactions within the GATOR complex

• Biomarker development: Evaluating WDR59 as a potential biomarker for mTORC1 pathway activity in disease states

• Tissue-specific interventions: Given WDR59's differential effects across tissues , these antibodies could help identify tissue-selective therapeutic approaches

• Combination therapy assessment: Analyzing how modulating WDR59 might enhance or reduce efficacy of existing mTOR inhibitors

This research direction could reveal new therapeutic strategies for diseases involving dysregulated mTOR signaling, such as cancer and metabolic disorders.

What emerging technologies could enhance the utility of WDR59 antibodies in investigating amino acid sensing mechanisms?

Several cutting-edge technologies could significantly advance WDR59 research:

• Single-cell protein analysis: Applying biotin-conjugated WDR59 antibodies in mass cytometry or single-cell Western blot technologies to assess cell-to-cell variability in WDR59 function

• Live-cell imaging: Developing membrane-permeable antibody fragments to track WDR59 dynamics in living cells during nutrient fluctuations

• CRISPR-based genetic screens: Using WDR59 antibodies to validate hits from genome-wide screens for amino acid sensing components

• Proximity labeling proteomics: Combining biotin-conjugated antibodies with BioID or APEX approaches to identify novel WDR59 interactors under different nutritional states

• Structural biology integration: Using antibodies to stabilize WDR59 complexes for cryo-EM structural studies of GATOR complexes

These technological advances could provide unprecedented insights into the molecular mechanisms of nutrient sensing and mTORC1 regulation.

How can contradictory findings about WDR59 function be reconciled through advanced experimental design?

The dual role of WDR59 in both promoting and inhibiting TORC1 activity presents an intriguing research puzzle . To reconcile these seemingly contradictory findings:

• Context-specific interaction mapping: Use biotin-conjugated antibodies in tissue-specific interactome studies to identify differential binding partners

• Post-translational modification analysis: Investigate how WDR59 modifications might switch its function between promotional and inhibitory roles

• Conditional knockout models: Develop tissue-specific and inducible WDR59 deletion systems to directly compare loss-of-function phenotypes across tissues

• Chimeric protein approaches: Create domain-swapping experiments to identify which regions confer inhibitory versus promotional functions

• Metabolomic integration: Correlate WDR59 activity with metabolic profiles to understand how cellular energy status might influence its function

These sophisticated experimental approaches could resolve the apparent paradox of WDR59's dual functionality, potentially revealing complex regulatory mechanisms that have evolved to fine-tune TORC1 signaling across different physiological contexts.