WDR24 Antibody, Biotin conjugated

What is WDR24 and what cellular pathways does it regulate?

WDR24 (WD Repeat Domain 24) functions as a critical component of the GATOR2 complex within the amino acid-sensing branch of the TORC1 signaling pathway. It acts as an activator of the TORC1 pathway by inhibiting the GATOR1 subcomplex, which itself inhibits this pathway . WDR24 is negatively regulated by upstream amino acid sensors including SESN2 and CASTOR1 . Beyond TORC1 regulation, WDR24 promotes lysosomal acidification and facilitates autophagic flux, establishing its dual role in cellular metabolism .

What are the key specifications of WDR24 Antibody, Biotin conjugated?

This antibody is specifically designed for enhanced detection sensitivity through biotin conjugation while maintaining high specificity for human WDR24 protein .

How should I optimize WDR24 antibody dilutions for different experimental applications?

While specific dilutions for the biotin-conjugated version may need to be determined empirically, related WDR24 antibodies have established working concentrations that can serve as starting points:

It's essential to titrate the antibody in your specific experimental system to achieve optimal results . For biotin-conjugated antibodies specifically, begin with manufacturer-recommended dilutions for ELISA applications and adjust based on signal-to-noise ratio.

What antigen retrieval methods are most effective for IHC applications with WDR24 antibodies?

For immunohistochemistry applications using WDR24 antibodies, the suggested antigen retrieval method is TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 can also be effective . The optimal method depends on tissue type, fixation protocol, and embedding medium. For formalin-fixed, paraffin-embedded tissues, heat-induced epitope retrieval is generally recommended. Consider performing a comparative analysis of both buffer systems to determine which provides the best signal-to-noise ratio for your specific experimental conditions.

How can I use WDR24 antibodies to investigate amino acid sensing mechanisms?

WDR24 plays a central role in amino acid-sensing pathways, making WDR24 antibodies valuable tools for studying nutrient regulation of cellular metabolism:

• Nutrient deprivation studies: Monitor WDR24 expression and localization during amino acid starvation and refeeding to assess dynamic regulation.

• GATOR complex interactions: Use co-immunoprecipitation with WDR24 antibodies to capture and analyze protein-protein interactions within the GATOR2 complex and between GATOR2 and GATOR1.

• Upstream sensor analysis: Investigate how amino acid sensors like SESN2 and CASTOR1 regulate WDR24 function by examining their interaction under varying nutrient conditions .

• Downstream effector activation: Assess how WDR24 levels correlate with mTORC1 activation by monitoring phosphorylation of downstream targets like S6K and 4E-BP1.

• Spatiotemporal regulation: Use immunofluorescence to track WDR24 localization relative to lysosomes and other organelles during nutrient fluctuations.

What methodological approaches can uncover WDR24's dual role in TORC1 signaling and autophagy?

WDR24's unique position at the intersection of nutrient sensing and autophagy regulation presents interesting research opportunities:

• Lysosomal acidification assays: Use lysosomal pH indicators (e.g., LysoTracker) alongside WDR24 immunofluorescence to correlate WDR24 levels with lysosomal function.

• Autophagic flux measurement: Monitor LC3-II and p62 levels in cells with normal, knocked-down, or overexpressed WDR24 to establish its contribution to autophagosome formation and clearance.

• Genetic manipulation approaches: Compare phenotypes of WDR24 knockout/knockdown with other GATOR2 components to distinguish between TORC1-dependent and independent functions .

• Rescue experiments: Introduce mutant forms of WDR24 with altered binding capacity to either GATOR1 or lysosomal proteins to dissect its bifunctional nature.

• Pharmacological inhibition studies: Assess how WDR24 function responds to compounds targeting either TORC1 (rapamycin) or autophagy (bafilomycin A1).

What are the most common technical issues when using WDR24 Antibody, Biotin conjugated in ELISA, and how can they be resolved?

For optimal results with biotin-conjugated antibodies, consider using streptavidin-based detection systems and including appropriate biotin blocking steps if endogenous biotin might be present in samples .

How can I validate the specificity of WDR24 antibody in my experimental system?

Rigorous validation is essential for ensuring reliable results with WDR24 antibodies:

• Positive controls: Include cell lines known to express WDR24, such as HEK-293, HeLa, or mouse brain tissue samples .

• Knockout/knockdown validation: Compare staining patterns between wild-type samples and those with reduced WDR24 expression through siRNA, shRNA, or CRISPR-Cas9 approaches .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to confirm that binding is specifically blocked.

• Multiple antibody comparison: Use antibodies targeting different epitopes of WDR24 to confirm consistent detection patterns.

• Molecular weight verification: Confirm that detected bands in Western blot appear at the expected molecular weights (102 kDa or 88 kDa) .

• Subcellular localization patterns: Verify that immunostaining patterns are consistent with known WDR24 localization.

How might WDR24 function in T cell immunity and nutrient sensing integration?

Recent CRISPR screens have implicated WDR24 in nutrient licensing of T cell immunity , suggesting several important research directions:

• T cell metabolic reprogramming: Investigate how WDR24 mediates the metabolic switch during T cell activation by comparing naïve, effector, and memory T cells.

• Immunometabolism crosstalk: Examine how WDR24-mediated amino acid sensing influences key T cell functions including cytokine production, proliferation, and effector functions.

• Nutrient availability and immune response: Assess how fluctuations in amino acid levels affect WDR24-dependent TORC1 signaling in T cells during infection or inflammation.

• Autophagy in immune homeostasis: Explore the connection between WDR24's role in autophagy and T cell survival, particularly in memory T cell formation and maintenance.

• Therapeutic targeting: Investigate whether modulation of WDR24 activity could enhance immunotherapy responses or mitigate autoimmunity by altering T cell metabolic programming.

What experimental approaches can elucidate WDR24's potential contributions to disease pathology?

WDR24's dual roles in TORC1 signaling and autophagy suggest its potential involvement in multiple disease processes:

• Cancer metabolism studies: Compare WDR24 expression and activity in tumor versus normal tissues to determine its role in cancer cell nutrient adaptation.

• Neurodegenerative disease models: Investigate whether WDR24 dysfunction contributes to autophagic defects observed in conditions like Alzheimer's and Parkinson's diseases.

• Lysosomal storage disorders: Examine WDR24's contribution to lysosomal acidification and function in models of lysosomal storage diseases.

• Metabolic disorders: Assess WDR24 activity in insulin-responsive tissues during obesity or diabetes to understand its role in metabolic dysregulation.

• Longitudinal expression analysis: Monitor changes in WDR24 levels during disease progression to identify potential windows for therapeutic intervention.

The biotin-conjugated WDR24 antibody offers particular advantages for multiplexed tissue analysis in complex disease models, allowing simultaneous detection of WDR24 alongside other biomarkers .