WDR59 Antibody, HRP conjugated

What is WDR59 and why is it important in cellular signaling research?

WDR59 is a component of the GATOR subcomplex GATOR2 that functions within the amino acid-sensing branch of the TORC1 signaling pathway. It indirectly activates mTORC1 and the TORC1 signaling pathway through inhibition of the GATOR1 subcomplex . The protein contains multiple tandem WD40 domains at the N-terminus arranged into β-propeller structure, and a putative C-terminal Ring domain . WDR59 is of significant research interest because:

• It plays a dual role in TORC1 regulation, functioning to either promote or inhibit TORC1 activity depending on cellular context

• It is negatively regulated by upstream amino acid sensors SESN2 and CASTOR1

• The Ring domains of WDR59 are essential for amino acid-mediated mTORC1 activation

• It serves as an important component in lysosomal recruitment of mTORC1 and related complexes

What are the key applications for WDR59 antibodies with HRP conjugation?

WDR59 antibodies with HRP conjugation are optimized for several detection methods:

The direct HRP conjugation provides advantages including elimination of secondary antibody incubation steps, reduced background, and increased detection sensitivity due to signal amplification properties of the HRP enzyme .

How should researchers store and handle WDR59 antibody with HRP conjugation to maintain activity?

Proper storage and handling are critical for maintaining the activity of HRP-conjugated antibodies:

• Store in light-protected vials or cover with a light-protecting material (aluminum foil)

• HRP-conjugated antibodies are stable for at least 12 months at 4°C

• For longer storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

• Avoid repeated freezing and thawing as this will compromise both enzyme activity and antibody binding

• Some formulations are supplied in high phosphate PBS (100 mM phosphate, 150 mM NaCl, pH 7.6)

• When aliquoting for long-term storage, use small volumes to minimize freeze-thaw cycles

How can researchers validate the specificity of a WDR59 antibody in their experimental system?

Validation of WDR59 antibody specificity should include multiple approaches:

• Positive controls: Use cell lines known to express WDR59, such as HEK293T or MCF7 cells, which have been confirmed to express WDR59 through BioGPS gene expression data

• Knockout/knockdown validation: Compare antibody reactivity in:

  • Wild-type cells

  • WDR59 CRISPR knockout cells

  • siRNA-mediated knockdown cells

• Blocking peptide experiment: Pre-incubate antibody with the immunizing peptide (available as catalog items AAP71988 or AAP71989 for some antibodies)

• Cross-reactivity assessment: Check specificity across species using sequence homology information:

  • Human (100%)

  • Mouse (93-100%)

  • Rat (93-100%)

  • Other species (varying degrees of homology)

• Multiple antibody comparison: Use antibodies targeting different epitopes of WDR59 (N-terminal region vs. C-terminal region) to confirm consistent detection patterns

What are optimal protocols for using WDR59 antibody (HRP conjugated) in Western blot applications?

For optimal Western blot results with HRP-conjugated WDR59 antibodies:

Sample preparation:

• Lyse cells in buffer containing protease inhibitors

• Include phosphatase inhibitors if studying WDR59 phosphorylation states

• Heat samples at 95°C for 5 minutes in SDS loading buffer

Electrophoresis and transfer:

• Run samples on 8-10% SDS-PAGE (WDR59 is approximately 110 kDa)

• Use wet transfer to PVDF membrane at 100V for 90 minutes

Blocking and antibody incubation:

• Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Dilute HRP-conjugated WDR59 antibody to 1:1000 in blocking solution

• Incubate overnight at 4°C or for 2 hours at room temperature

• Wash 3-5 times with TBST (5 minutes each wash)

Detection:

• Develop using enhanced chemiluminescence (ECL) substrate

• Expected band size: approximately 110 kDa

• Note: Since no secondary antibody is needed, proceed directly to detection after washing

How can researchers troubleshoot weak or no signal when using WDR59 antibody with HRP conjugation?

When facing detection issues with HRP-conjugated WDR59 antibodies, consider these troubleshooting approaches:

Problem: Weak or no signal

• Cause: Insufficient antigen, enzyme inactivation, or inappropriate detection conditions

• Solutions:

  • Increase protein loading (25-50 μg total protein recommended)

  • Decrease antibody dilution (use more concentrated antibody)

  • Extend exposure time during detection

  • Verify HRP activity using a direct enzyme activity assay

  • Use fresh ECL substrate and ensure proper working condition of imaging equipment

  • Check if target cells/tissues express sufficient WDR59 (reference expression databases)

Problem: High background

• Cause: Non-specific binding or inappropriate blocking

• Solutions:

  • Optimize blocking conditions (try BSA instead of milk or vice versa)

  • Increase washing duration and number of washes

  • Dilute antibody further

  • Ensure all buffers are freshly prepared

Problem: Multiple bands

• Cause: Degradation, splice variants, or cross-reactivity

• Solutions:

  • Use fresh sample preparation with additional protease inhibitors

  • Verify expected molecular weight (approximately 110 kDa for WDR59)

  • Consider using a different WDR59 antibody targeting a different epitope to confirm specificity

How can researchers use WDR59 antibody (HRP conjugated) to investigate the differential roles of WDR59 in TORC1 regulation?

WDR59 exhibits context-dependent functions in TORC1 regulation, acting as either an activator or inhibitor depending on cellular environment . To investigate these differential roles:

Experimental approach:

• Cell-type specific analysis:

  • Compare WDR59 expression and localization across multiple cell types using immunofluorescence and Western blot

  • Correlate WDR59 localization with TORC1 activity using phospho-S6K detection

• Amino acid starvation and refeeding experiments:

  • Subject cells to amino acid starvation (60 minutes in amino acid-free RPMI)

  • Examine WDR59 localization and interaction partners before and after amino acid readdition

  • Monitor changes in TORC1 activity markers (phospho-S6K, phospho-S6, phospho-4EBP1)

• Subcellular fractionation:

  • Isolate lysosomal fractions using lysosomal immunocapture techniques

  • Compare WDR59 recruitment to lysosomes under different nutrient conditions

  • Analyze co-recruitment of other GATOR complex components

• Co-immunoprecipitation studies:

  • Use anti-WDR59 antibodies to pull down protein complexes under different conditions

  • Analyze interaction partners through mass spectrometry

  • Compare GATOR1 vs. GATOR2 associations in different cellular contexts

• Tissue-specific knockout models:

  • Generate conditional WDR59 knockout in different tissues

  • Analyze phenotypes related to cell growth and autophagy

  • Correlate with TORC1 activity measurements

This comprehensive approach will help elucidate how WDR59 switches between promoting and inhibiting TORC1 activity in different contexts .

What methods are effective for studying WDR59 interactions with other components of the amino acid sensing pathway using HRP-conjugated antibodies?

To investigate WDR59 interactions within the amino acid sensing pathway:

Co-immunoprecipitation approaches:

• Use HRP-conjugated WDR59 antibody for immunoblotting after standard IP with non-conjugated antibodies against interaction partners

• Investigate interactions with:

  • Other GATOR2 components (WDR24, Mios, Sec13, Seh1L)

  • GATOR1 components (NPRL2, NPRL3, DEPDC5)

  • Rag GTPases

  • Amino acid sensors (SESN2, CASTOR1)

Proximity-based protein interaction assays:

• Implement NanoBit-based interaction assays to monitor Ring-Ring domain interactions between WDR59, WDR24, and Mios

• Use this approach to analyze how mutations in conserved residues affect binding affinity

Immunofluorescence co-localization:

• Perform dual immunofluorescence using:

  • Anti-WDR59 antibody

  • Markers for lysosomes (LAMP2)

  • Other GATOR complex components

• Analyze co-localization under different nutrient conditions

• Use Alexa488-conjugated secondary antibody for WDR59 staining

Functional assays to measure GATOR2 activity:

• Monitor NPRL2 ubiquitination levels which are physiologically regulated in an amino acid-sensitive manner

• Investigate how WDR59 affects this ubiquitination process

• Correlate with measurements of mTORC1 activity

These approaches provide a comprehensive framework for understanding WDR59's role in the complex network of amino acid sensing and mTORC1 regulation.

How can researchers investigate the functional significance of WDR59 Ring domain using antibodies that target different regions of the protein?

The Ring domain of WDR59 is essential for amino acid-mediated mTORC1 activation . To investigate its functional significance:

Domain-specific antibody approach:

• Use antibodies targeting different regions of WDR59:

  • N-terminal region antibodies (such as ARP71988-P050-HRP)

  • C-terminal region antibodies (such as ARP71989-P050-HRP)

  • Ring domain-specific antibodies (AA 619-862)

• CRISPR-mediated domain mutation experiments:

  • Generate cells with CRISPR/Cas9-mediated knock-in of Ring domain mutations (e.g., C743A/C746A)

  • Use antibodies to confirm expression of mutant proteins

  • Compare localization and complex formation capability of wild-type vs. mutant WDR59

• Structure-function analysis workflow:

  • Express full-length WDR59 vs. truncated versions lacking Ring domain

  • Analyze impact on:

    • GATOR2 complex integrity

    • Interaction with GATOR1

    • mTORC1 activation (phospho-S6K, phospho-4EBP1)

    • Cell growth phenotypes

• Ring domain interaction studies:

  • Use in vitro GST pull-down assays to analyze Ring-Ring domain interactions

  • Implement NanoBit-based interaction assays to monitor these interactions in cells

  • Evaluate how mutations in conserved residues affect binding affinity

• Functional rescue experiments:

  • In WDR59-depleted cells, reintroduce either wild-type or Ring domain mutant WDR59

  • Assess ability to restore normal mTORC1 signaling and amino acid responsiveness

  • Monitor interactions with other GATOR complex components

This comprehensive approach will help elucidate the specific role of the Ring domain in WDR59 function and its impact on mTORC1 signaling.

What are the methodological considerations for using WDR59 antibodies to study tissue-specific functions in developmental or disease models?

When investigating WDR59 in tissue-specific contexts:

Tissue processing and fixation:

• For immunohistochemistry with HRP-conjugated WDR59 antibodies:

  • Use formalin/PFA-fixed paraffin-embedded sections

  • Optimal dilution range: 1:20-1:50

  • Include antigen retrieval steps (citrate buffer pH 6.0, 95°C for 20 minutes)

  • Expected result: Strong cytoplasmic positivity in certain cell types (e.g., glandular cells in thyroid)

Disease model considerations:

• Cancer models:

  • WDR59 expression has been identified in MCF7 breast cancer cells

  • Compare expression and localization between normal and malignant tissues

  • Correlate with mTORC1 activity and cell growth phenotypes

• Developmental models:

  • Study WDR59 in Drosophila female germline where it functions as a TORC1 inhibitor

  • Compare with other tissues where it may function as an activator

  • Use genetic tools to create tissue-specific knockouts

• Nutrient sensing experiments:

  • Compare WDR59 function in nutrient-responsive tissues (liver, muscle) vs. other tissues

  • Study response to feeding/fasting cycles

  • Analyze colocalization with lysosomes (LAMP2) in different physiological states

• Technical validation:

  • Include appropriate positive controls based on known expression patterns

  • Use gene expression databases to guide tissue selection

  • Validate antibody specificity in each tissue type studied

  • Consider species cross-reactivity when working with animal models:

    • Human (100% homology to immunogen)

    • Mouse (93-100% homology)

    • Rat (93-100% homology)

    • Other species (varying degrees of homology)

This methodological framework enables robust investigation of WDR59's tissue-specific functions in various biological contexts.