WDR41 Antibody, Biotin conjugated

What applications is the WDR41 Antibody, Biotin conjugated suitable for?

The WDR41 Antibody, Biotin conjugated has been validated primarily for ELISA applications according to manufacturer specifications . Unlike its unconjugated counterpart, which has demonstrated utility in Western Blot applications with a recommended dilution range of 1:500-1:3000, the biotin-conjugated version's applications are more specialized . The biotin conjugation provides advantages in detection systems utilizing streptavidin, which can enhance signal amplification in immunoassays . Researchers should note that application suitability may be sample-dependent, and optimization through antibody titration is recommended for each specific experimental system to obtain optimal results .

How should WDR41 Antibody, Biotin conjugated be stored and handled to maintain its activity?

Proper storage of WDR41 Antibody, Biotin conjugated is crucial for maintaining its activity and specificity. The manufacturer recommends storing the antibody at -20°C or -80°C upon receipt . The antibody should be stored in its buffer composition consisting of 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . Researchers should avoid repeated freeze-thaw cycles as these can compromise antibody integrity and activity . For optimal results, aliquoting the antibody before storage is advisable, though the manufacturer notes this is unnecessary for -20°C storage for the smaller 20μl size which contains 0.1% BSA . When handling, maintain sterile conditions and avoid contamination to preserve antibody performance in experimental applications.

How does WDR41 interact with the C9orf72-SMCR8 complex, and what are the functional implications?

WDR41 exhibits a specific binding pattern within the C9orf72-SMCR8 complex through distinct structural domains. Cryo-EM structural analysis reveals that WDR41 primarily interacts with SMCR8 via its C-terminal helix (WDR41 CTH) and N-terminal β-strand without direct physical contact with C9orf72 . Specifically, WDR41 CTH packs against a groove formed by αD11, αD12, and αD13 of SMCR8, while the N-terminal β-strand of WDR41 forms an antiparallel β-sheet with βD6 of SMCR8 . Experimentally, deletion of WDR41's C-terminal helix (residues 436-459) abolishes its interaction with the C9orf72-SMCR8 complex, confirming the critical importance of this region .

The functional significance of this interaction is substantial, as WDR41 is essential for the localization of the C9orf72-SMCR8 complex to lysosomes . Analysis of WDR41 knockout cells demonstrated that WDR41 is required for the lysosomal localization of this complex, particularly in response to amino acid starvation . This recruitment mechanism is independent of mTORC1 inhibition or autophagy induction . Importantly, WDR41-dependent recruitment of C9orf72 to lysosomes is critical for the ability of lysosomes to support mTORC1 signaling, as demonstrated by experiments showing that constitutive targeting of C9orf72 to lysosomes relieves the requirement for WDR41 in mTORC1 activation .

What are the technical considerations when using WDR41 Antibody, Biotin conjugated for colocalization studies?

When designing colocalization experiments using WDR41 Antibody, Biotin conjugated, researchers should carefully consider several technical aspects to ensure reliable results. First, the subcellular localization of WDR41 is dynamic and condition-dependent - while WDR41 shows diffuse staining under normal growth conditions, it significantly colocalizes with LAMP1 (a lysosomal marker) in response to starvation . This condition-dependent localization requires careful experimental design with appropriate positive and negative controls.

For detection systems, researchers should utilize streptavidin conjugated to fluorophores that are spectrally distinct from other fluorophores used in the experiment to avoid bleed-through . When studying WDR41's lysosomal localization, researchers should employ parallel methodologies for verification - both immunofluorescence and biochemical approaches (such as magnetic purification of lysosomes followed by immunoblotting) have proven effective in confirming WDR41's lysosomal enrichment .

Importantly, overexpression studies may produce misleading results; previous research using overexpressed GFP-tagged WDR41 reported Golgi localization, while analysis of endogenously expressed protein revealed lysosomes as the major site of enrichment . Therefore, working with endogenous protein levels or using CRISPR/Cas9-mediated genome editing to insert tags (such as HA) at the endogenous locus provides more physiologically relevant localization data .

How can researchers effectively analyze WDR41 mutations and their impact on complex formation?

When designing mutation studies, researchers should consider both individual residue mutations and combinations affecting key structural elements. For functional validation of mutations, pulldown assays with recombinant proteins provide a reliable method to assess complex formation . Additionally, researchers should validate the impact of mutations in cellular contexts through co-immunoprecipitation experiments, which can reveal if complex formation is disrupted in a cellular environment .

For comprehensive analysis, researchers should combine structural approaches (examining how mutations affect protein folding and complex assembly) with functional assays (determining how mutations impact WDR41's ability to recruit the C9orf72-SMCR8 complex to lysosomes or affect mTORC1 signaling) . This multi-faceted approach will provide deeper insights into structure-function relationships of WDR41 and its role within the complex.

What are the recommended protocols for detecting WDR41 in subcellular fractions?

Detection of WDR41 in subcellular fractions requires carefully optimized protocols that account for its dynamic localization patterns. For lysosomal fraction analysis, magnetic immunoisolation has proven effective - researchers successfully used this approach to confirm WDR41's starvation-induced lysosomal localization . This technique involves using magnetic beads coated with antibodies against lysosomal membrane proteins to isolate intact lysosomes, followed by immunoblotting with anti-WDR41 antibodies .

For immunofluorescence detection of endogenous WDR41, CRISPR/Cas9-mediated tagging (e.g., with 2xHA) is recommended over antibody-based detection due to validation challenges with available antibodies for this application . When studying WDR41's dynamic localization, researchers should compare both fed and starved conditions, as WDR41 shows marked recruitment to lysosomes specifically during starvation . A standard starvation protocol involves incubating cells in EBSS (Earle's Balanced Salt Solution) without amino acids for 2-4 hours before fixation or fractionation .

For Western blot analysis using biotin-conjugated antibodies, a dilution range starting from 1:500-1:3000 is recommended based on data from related WDR41 antibodies . Optimization of blocking conditions is critical - 5% BSA in TBST is generally effective for minimizing background with biotin-conjugated antibodies . Detection can be performed using streptavidin-HRP with enhanced chemiluminescence for sensitive visualization of WDR41, which typically appears at approximately 52 kDa on immunoblots .

How can researchers validate specificity of WDR41 Antibody, Biotin conjugated in their experimental system?

Validation of WDR41 Antibody, Biotin conjugated specificity is essential for reliable experimental outcomes. A comprehensive validation approach should include multiple complementary strategies. First, researchers should perform Western blot analysis comparing wild-type samples with WDR41 knockout controls generated using CRISPR/Cas9 technology . Previous studies have successfully created and validated WDR41 knockout cell lines in HeLa and HEK293FT backgrounds, which serve as excellent negative controls .

For immunoprecipitation experiments, researchers should verify that the antibody pulls down a protein of the expected molecular weight (52 kDa for WDR41) and confirm its identity through mass spectrometry . Additionally, peptide competition assays can be performed using the immunogen - recombinant Human WD repeat-containing protein 41 (amino acids 7-284) - to demonstrate specific binding that can be competitively inhibited .

When using the antibody for ELISA applications, researchers should establish standard curves using purified recombinant WDR41 protein at known concentrations and demonstrate linear response within the working range . Cross-reactivity testing against related WD-repeat proteins can further confirm specificity. For additional validation, researchers can compare results obtained with the biotin-conjugated antibody against those obtained with other validated anti-WDR41 antibodies targeting different epitopes .

What technical approaches can address challenges in studying WDR41 interactions in neurodegenerative disease models?

Studying WDR41 in neurodegenerative disease contexts presents unique challenges that require specialized approaches. Since WDR41 has been implicated in ALS and FTD through its interaction with C9orf72 , researchers should consider both cellular and animal models that recapitulate disease-relevant mutations. For C9orf72 hexanucleotide repeat expansion models, researchers can use CRISPR/Cas9 to introduce the expansion into cell lines or use patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons .

When examining protein interactions, proximity ligation assays (PLA) offer advantages over conventional co-immunoprecipitation by detecting protein interactions in situ with subcellular resolution . This approach is particularly valuable for studying WDR41's dynamic interactions with C9orf72 and SMCR8 under different cellular stresses relevant to neurodegeneration . For detecting subtle changes in complex formation or localization, super-resolution microscopy techniques such as STORM or STED provide the necessary spatial resolution to distinguish lysosomal recruitment patterns .

To address the challenge of low protein expression in neuronal models, researchers can employ more sensitive detection methods such as Tyramide Signal Amplification (TSA) with the biotin-conjugated antibody . For functional studies, CRISPR interference (CRISPRi) or inducible knockdown systems allow for temporal control of WDR41 depletion, which can help distinguish between developmental and acute effects of WDR41 dysregulation in neuronal systems . Lastly, mass spectrometry-based interactomics comparing wild-type and disease model conditions can reveal alterations in the WDR41 interactome that may contribute to disease pathogenesis .

How should researchers interpret changes in WDR41 localization during cellular stress responses?

When interpreting these localization changes, researchers should consider that WDR41's lysosomal recruitment occurs in parallel with, and is functionally connected to, C9orf72 recruitment to lysosomes . This coordinated recruitment is specifically responsive to amino acid availability rather than general stress responses, as it is not dependent on mTORC1 inhibition or autophagy induction . Therefore, observed changes in WDR41 localization should be interpreted primarily as indicators of amino acid sensing mechanisms rather than general autophagy activation.

Importantly, contrary to earlier reports suggesting Golgi localization based on overexpressed GFP-tagged constructs, studies of endogenously expressed WDR41 firmly establish lysosomes as the major site of WDR41 enrichment under starvation conditions . This highlights the critical importance of studying proteins at endogenous levels or using minimally disruptive tagging strategies when interpreting localization data . Researchers should also note that WDR41's lysosomal recruitment has functional consequences for mTORC1 signaling, making it an important readout for assessing the functionality of nutrient sensing pathways .

How can researchers reconcile conflicting findings about WDR41's role in different experimental systems?

Reconciling conflicting findings about WDR41 requires careful consideration of several experimental variables. First, differences in localization findings (such as Golgi versus lysosomal localization) can often be attributed to expression levels and tagging strategies . Overexpression systems typically used in earlier studies may lead to mislocalization, while more recent approaches examining endogenously expressed or endogenously tagged WDR41 provide more physiologically relevant localization data . Therefore, researchers should prioritize findings from experimental systems that maintain endogenous expression levels.

Second, cell type-specific differences should be considered when evaluating WDR41 function. While core interactions with C9orf72 and SMCR8 appear consistent across cell types, the downstream effects on pathways like autophagy and mTORC1 signaling may vary based on cell-specific contexts . When comparing studies, researchers should explicitly note the cell types used and consider how metabolic differences between cell types might influence nutrient sensing functions of WDR41.

Additionally, experimental conditions - particularly nutrient availability - dramatically affect WDR41 localization and function . Studies conducted under different nutritional states (fed versus starved) may yield apparently conflicting results that actually reflect the dynamic nature of WDR41's function . Temporal considerations are also important; acute versus chronic manipulations of WDR41 may produce different phenotypes due to compensatory mechanisms.

To reconcile conflicting findings, researchers should perform side-by-side comparisons using standardized experimental conditions and multiple complementary approaches (biochemical, imaging, and functional assays) to build a more coherent understanding of WDR41's context-dependent functions .

What are the promising therapeutic implications of targeting WDR41 in neurodegenerative diseases?

The connection between WDR41 and neurodegenerative diseases, particularly ALS and FTD, offers intriguing therapeutic possibilities . Since C9orf72 mutations represent a major cause of these conditions, and WDR41 is essential for proper C9orf72 complex function and localization, targeting WDR41 could potentially modulate disease processes . The structural details now available for the C9orf72-SMCR8-WDR41 complex provide molecular targets for developing small molecules that could enhance or inhibit specific interactions within the complex .

Potential therapeutic strategies could aim to restore proper lysosomal localization of the C9orf72 complex in disease states where this process is disrupted . Since WDR41-dependent recruitment of C9orf72 to lysosomes is critical for mTORC1 signaling, compounds that enhance this recruitment might ameliorate defects in nutrient sensing pathways that contribute to neurodegeneration . Conversely, in contexts where excessive autophagy inhibition contributes to pathology, inhibiting the interaction between WDR41 and SMCR8 could potentially reduce the complex's ability to negatively regulate autophagy initiation .

The identification of specific residues critical for WDR41-SMCR8 interaction, such as L445, F446, and L449 on WDR41's C-terminal helix, provides precise targets for rational drug design . Peptide-based therapeutics mimicking the C-terminal helix of WDR41 could potentially modulate the formation or function of the complex in disease contexts .

As research in this area progresses, researchers should focus on developing tools to monitor WDR41 function in living neurons and create more sophisticated disease models to test the effects of WDR41 manipulation on disease progression and pathology .

What methodological advances are needed to better study WDR41 in primary neurons and brain tissue?

Studying WDR41 in primary neurons and brain tissue presents unique challenges that require methodological innovations. Current antibodies, including the biotin-conjugated version, have been primarily validated in cell lines rather than in neuronal contexts . Developing and rigorously validating antibodies specifically for neuronal immunohistochemistry and brain tissue analysis represents an important methodological advance needed in this field.

For in vivo studies, generating neuron-specific conditional WDR41 knockout mouse models would allow researchers to investigate the consequences of WDR41 loss in intact neural circuits . Additionally, developing AAV-based tools for regional and cell-type-specific manipulation of WDR41 expression in adult brains would enable more precise temporal control over WDR41 function in specific neural populations .

To study the dynamic localization of WDR41 in living neurons, researchers need to develop minimally disruptive fluorescent tagging strategies compatible with neuronal physiology . CRISPR-mediated knock-in of small fluorescent tags at the endogenous WDR41 locus in neuronal models would allow real-time visualization of WDR41 trafficking in response to various physiological stimuli and stressors .

For biochemical studies in brain tissue, optimizing methods for isolating intact lysosomes from specific brain regions would enable region-specific analysis of WDR41's lysosomal recruitment in health and disease models . Additionally, developing effective extraction protocols for membrane-associated protein complexes from brain tissue would facilitate more accurate analysis of the C9orf72-SMCR8-WDR41 complex in its native neuronal environment .

Lastly, establishing standardized protocols for culturing and analyzing patient-derived neurons from individuals with C9orf72 mutations would provide a more disease-relevant context for studying WDR41 function in human neurons .