WDR24 Antibody, HRP conjugated

What is WDR24 and why is it significant in cellular research?

WDR24 is a critical component of the GATOR2 complex involved in multiple cellular processes including protein degradation, gene expression regulation, and cell cycle progression. The protein contains approximately 920 amino acids with a calculated molecular weight of 102 kDa, though it is often observed at 88-102 kDa in experimental conditions . WDR24's significance lies primarily in its role in the mTORC1 signaling pathway, particularly in amino acid sensing mechanisms. Research has demonstrated that WDR24 possesses intrinsic E3 ubiquitin ligase activity mediated through its Ring domain structure, which is essential for proper mTORC1 activation in response to amino acid stimulation .

The protein's involvement in these fundamental pathways makes it a valuable target for studying cellular nutrient sensing, growth regulation, and potential connections to diseases where these pathways are dysregulated.

Detection of WDR24 can be accomplished through either direct conjugation of labels to anti-WDR24 antibodies or using unconjugated primary antibodies followed by labeled secondary antibodies.

In direct detection, the anti-WDR24 antibody itself carries the detection molecule (such as HRP). This approach offers faster protocols with fewer washing steps and reduced cross-reactivity, but often provides lower sensitivity due to limited signal amplification.

For indirect detection, an unconjugated anti-WDR24 primary antibody (typically raised in rabbit, as seen with products like 20778-1-AP) is first applied, followed by an HRP-conjugated secondary antibody that recognizes rabbit IgG . This method provides signal amplification as multiple secondary antibodies can bind each primary antibody, enhancing sensitivity. The recommended approach for Western blotting involves diluting anti-WDR24 primary antibodies between 1:500-1:2000, followed by appropriate HRP-conjugated secondary antibodies at 1:5000-1:100000 dilution .

How should sample preparation be optimized for WDR24 Western blot analysis?

Effective Western blot detection of WDR24 requires careful sample preparation:

• Cell lysis should be performed using buffers containing protease inhibitors to prevent degradation of WDR24.

• For optimal separation, use 5-20% gradient SDS-PAGE gels and run at 70V (stacking) followed by 90V (resolving) for 2-3 hours .

• Load approximately 30 μg of protein per lane under reducing conditions.

• Transfer proteins to nitrocellulose membranes at 150 mA for 50-90 minutes.

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature.

• Incubate with anti-WDR24 antibody (e.g., A12216 at 0.5 μg/mL) overnight at 4°C.

• Wash with TBS containing 0.1% Tween three times (5 minutes each).

• Probe with HRP-conjugated secondary antibody (typically anti-rabbit IgG) at 1:5000 dilution for 1.5 hours.

• Develop using enhanced chemiluminescent detection.

WDR24 should appear as a specific band at approximately 88-102 kDa, though the exact molecular weight may vary between tissues and cell lines .

What controls are essential for validating WDR24 antibody specificity?

Validating WDR24 antibody specificity requires several controls:

• Positive controls: Use known WDR24-expressing samples such as HEK-293 cells, HeLa cells, or human placenta tissue lysates .

• Negative controls:

  • Primary antibody omission

  • CRISPR/Cas9 WDR24 knockout cells or knockdown samples

  • Competing peptide blocking studies

• Cross-reactivity assessment: Test the antibody against multiple species samples if cross-reactivity is claimed (human, mouse, and rat samples are typically reactive) .

• Molecular weight verification: Confirm that the observed molecular weight matches the expected size (88-102 kDa for WDR24) .

Multiple validation methods should be employed to establish confidence in antibody specificity, particularly for critical research applications .

How can immunoprecipitation be optimized for studying WDR24 protein interactions?

For effective WDR24 immunoprecipitation:

• Use 0.5-4.0 μg of anti-WDR24 antibody per 1.0-3.0 mg of total protein lysate .

• Consider using CRISPR-Cas9 system to insert an in-frame Flag-tag at the endogenous WDR24 gene locus to ensure physiological immunoprecipitation of the GATOR2 complex .

• For studying protein interactions, perform co-immunoprecipitation under gentle lysis conditions that preserve protein-protein interactions.

• When studying amino acid-dependent interactions, compare immunoprecipitation results from amino acid-starved versus amino acid-stimulated conditions .

• Size-exclusion chromatography (SEC) can be combined with immunoprecipitation to analyze the intact GATOR2 complex and its interactions with GATOR1 .

• For detecting ubiquitination, particularly of NPRL2 by WDR24, include deubiquitinase inhibitors in lysis buffers and consider using tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins .

How can WDR24 antibodies be used to study Ring domain function in mTORC1 signaling?

The Ring domain of WDR24 plays a crucial role in mTORC1 signaling and can be studied using specialized antibody applications:

• Domain-specific antibodies: Use antibodies that specifically recognize the Ring domain to monitor conformational changes upon amino acid stimulation.

• Mutational analysis: Compare antibody reactivity between wild-type WDR24 and Ring domain mutants (especially CA mutations that disrupt critical residues) to assess structural integrity .

• Proximity ligation assays: Combine WDR24 antibodies with antibodies against other GATOR2 components to study Ring-Ring domain interactions in situ.

• NanoBit-based interaction assays: Monitor Ring-Ring domain interactions using complementary antibodies to assess binding affinity in cells .

• Subcellular localization: Track WDR24 localization to lysosome surfaces in response to amino acid stimulation using immunofluorescence, as disruption of the Ring domain leads to defective mTORC1 localization .

• Ubiquitination activity: Monitor WDR24's E3 ubiquitin ligase activity by detecting ubiquitination of NPRL2 (the catalytic subunit of GATOR1) in response to amino acid stimulation, which is dependent on the Ring domain .

What methodologies can detect WDR24-mediated ubiquitination of target proteins?

To study WDR24's E3 ubiquitin ligase activity and substrate ubiquitination:

• In vivo ubiquitination assays: Transfect cells with HA-tagged ubiquitin, immunoprecipitate potential substrates (particularly NPRL2), and probe with anti-HA antibody to detect ubiquitin chains.

• In vitro ubiquitination assays: Purify WDR24 (wild-type and Ring domain mutants) and assess its ability to generate ubiquitin chains in the presence of E1 and E2 enzymes .

• Amino acid response monitoring: Compare ubiquitination levels of NPRL2 before and after amino acid stimulation, which physiologically regulates NPRL2 ubiquitination in an amino acid-sensitive manner .

• WDR24 dependency tests: Deplete WDR24 using siRNA or CRISPR-Cas9 and observe the effect on substrate ubiquitination. Re-expressing wild-type WDR24, but not Ring domain mutants, should restore ubiquitination .

• Proteasome inhibition: Treat cells with proteasome inhibitors (MG132, Bortezomib, or TAK-243) to accumulate ubiquitinated proteins and facilitate detection .

The ubiquitination of NPRL2 specifically occurs upon amino acid stimulation without affecting protein stability or the integrity of the GATOR1 complex, suggesting a regulatory rather than degradative role .

How do different fixation and permeabilization methods affect WDR24 immunofluorescence results?

The choice of fixation and permeabilization methods significantly impacts WDR24 immunofluorescence results:

For WDR24 immunofluorescence, optimization is essential as the protein functions both at lysosomes and in the cytosol depending on cellular conditions. When studying amino acid-dependent localization of WDR24 to lysosomes, minimizing fixation time and using gentle permeabilization (0.1% Triton X-100 or 0.05% saponin) helps preserve delicate membrane structures .

What factors contribute to inconsistent WDR24 Western blot results?

Several factors can lead to inconsistent WDR24 detection:

• Protein degradation: WDR24 protein levels are regulated post-translationally through ubiquitination. Depletion of Mios (another GATOR2 component) dramatically reduces WDR24 protein levels through enhanced ubiquitination and proteasomal degradation .

• Antibody selection: Different antibodies (A12216 vs. 20778-1-AP) may recognize different epitopes, leading to variability in detection sensitivity.

• Molecular weight variability: WDR24 appears at both 88 kDa and 102 kDa depending on post-translational modifications and experimental conditions .

• Sample preparation: Inadequate protease inhibition can lead to degradation products and inconsistent banding patterns.

• Transfer efficiency: Large proteins like WDR24 require optimized transfer conditions; insufficient transfer times can cause weak signal.

• Amino acid status: Since WDR24 function and possibly stability are regulated by amino acid availability, the nutritional status of cells before lysis can affect results .

To address these issues, standardize sample collection, use fresh protease inhibitors, optimize transfer conditions for larger proteins, and consider the amino acid status of experimental samples.

How can background be reduced in WDR24 immunohistochemistry applications?

To reduce background in WDR24 immunohistochemistry:

• Optimize antibody dilution: Test a range of dilutions between 1:50-1:500 to find the optimal signal-to-noise ratio .

• Antigen retrieval: Use TE buffer pH 9.0 as the primary recommendation for WDR24, though citrate buffer pH 6.0 may also be effective for some tissue types .

• Blocking optimization: Extend blocking time (1-2 hours) with appropriate blocking solution (5% normal serum from the same species as the secondary antibody).

• Secondary antibody concentration: Reduce concentration if high background persists.

• Endogenous peroxidase quenching: For HRP-based detection, thoroughly quench endogenous peroxidase activity with 3% hydrogen peroxide before antibody incubation.

• Use biotin-free detection systems: Consider polymer-based detection systems if streptavidin-biotin methods show high background.

• Washing optimization: Increase washing duration and volume between antibody incubations.

For human tissues specifically, WDR24 antibody has been validated for colon cancer tissue, colon tissue, and ovary tissue, showing specific cellular staining patterns .

How should changes in WDR24 ubiquitination activity be interpreted in amino acid signaling studies?

When interpreting WDR24 ubiquitination activity:

• Substrate specificity: WDR24 specifically ubiquitinates NPRL2 (the catalytic subunit of GATOR1), but not NPRL3 or DEPDC5, indicating a targeted regulatory mechanism .

• Amino acid sensitivity: Ubiquitination of NPRL2 increases upon amino acid stimulation without affecting protein stability or GATOR1 complex integrity, suggesting a non-degradative signaling role .

• Ring domain dependency: Wild-type WDR24, but not Ring domain mutants, can restore NPRL2 ubiquitination in WDR24-depleted cells, confirming the functional significance of this domain .

• GATOR2 complex integrity: Complete deletion of Mios or its Ring domain blunts amino acid-induced NPRL2 ubiquitination, highlighting the importance of intact GATOR2 for WDR24 function .

• mTORC1 regulation: Changes in WDR24-mediated ubiquitination correlate with mTORC1 localization to lysosomal surfaces, providing a mechanistic link between these events .

These findings collectively suggest that WDR24's ubiquitination activity serves as a regulatory switch in amino acid sensing pathways rather than targeting proteins for degradation.

What experimental approaches can determine if WDR24 antibody results are specific and reliable?

To validate WDR24 antibody specificity:

• Genetic knockdown/knockout validation: Use siRNA knockdown or CRISPR-Cas9 knockout of WDR24 to confirm signal reduction or loss with the antibody.

• Multiple antibody concordance: Compare results from different antibodies targeting distinct WDR24 epitopes (such as A12216 and 20778-1-AP) .

• Overexpression validation: Express tagged WDR24 and confirm co-localization with antibody staining.

• Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate specific blocking of signal.

• Cross-species reactivity: Confirm expected patterns in multiple species when cross-reactivity is claimed (human, mouse, rat) .

• Molecular weight verification: Confirm detection at the expected molecular weights (88-102 kDa) .

• Publication concordance: Compare results with published data using the same or different antibodies.

A comprehensive validation strategy helps ensure reliable interpretation of results, especially in complex signaling pathways involving WDR24.

How can researchers utilize WDR24 antibodies to explore the relationship between GATOR complexes and mTORC1 signaling?

WDR24 antibodies provide valuable tools for exploring GATOR-mTORC1 regulatory mechanisms:

• Subcellular co-localization studies: Use immunofluorescence to track WDR24 localization to lysosomes upon amino acid stimulation, which is critical for mTORC1 activation .

• Protein interaction networks: Apply co-immunoprecipitation with WDR24 antibodies to map interactions with both GATOR1 (NPRL2, NPRL3, DEPDC5) and GATOR2 components (Mios, Sec13, Seh1L, WDR59) .

• Structure-function analysis: Compare wild-type WDR24 with Ring domain mutants to assess the impact on mTORC1 activity, measuring phosphorylation of downstream targets like S6K and 4E-BP1.

• Nutrient response dynamics: Monitor WDR24 protein levels, localization, and interactions under various nutrient conditions to understand its role as a nutrient sensor.

• Ubiquitination cascade mapping: Use WDR24 antibodies in combination with ubiquitin antibodies to track the amino acid-induced ubiquitination of NPRL2 and its effect on GATOR1 inhibitory function .

• Pathological relevance: Examine WDR24 expression and function in cancer tissues where mTORC1 signaling is dysregulated, potentially revealing new therapeutic targets.

These applications of WDR24 antibodies contribute to our understanding of how cells sense and respond to nutrient availability through the intricate regulation of the mTORC1 pathway.