WDR83 Antibody, Biotin conjugated

What is WDR83 and why is it a target of study in research?

WDR83, also known as MORG1 (mitogen-activated protein kinase organizer 1), is a member of the WD-40 protein family that contains WD repeat domains. It functions in mitogen-activated protein kinase signaling pathways and has been implicated in various cellular processes. WDR83 has a calculated molecular weight of approximately 34.3 kDa, although it may appear at approximately 12 kDa in some experimental conditions . The UniProt ID for human WDR83 is Q9BRX9, which can be used to obtain detailed sequence and structural information about this protein .

What defines a biotin-conjugated WDR83 antibody?

A biotin-conjugated WDR83 antibody is an immunoglobulin that specifically recognizes WDR83 protein and has been chemically linked to biotin molecules. The biotin conjugation is typically achieved through a chemical reaction that couples biotin to primary amines on the antibody while preserving the antibody's antigen-binding ability. The resulting conjugate enables detection through the biotin-(strept)avidin interaction system, which is one of the strongest non-covalent interactions in nature (Kd ~10^-15 M), approximately 10^3 to 10^6 times stronger than a typical antigen–antibody interaction .

What are the key specifications to consider when selecting a biotin-conjugated WDR83 antibody?

When selecting a biotin-conjugated WDR83 antibody, researchers should consider:

• Host species: Typically rabbit for polyclonal antibodies against WDR83

• Clonality: Polyclonal or monoclonal (polyclonal is more common for WDR83)

• Reactivity: Species cross-reactivity (human, mouse, rat)

• Applications validated: ELISA, Western blot, immunohistochemistry, etc.

• Biotin conjugation method: Direct conjugation or spacer-incorporated biotin (e.g., Biotin-SP)

• Degree of biotinylation: Number of biotin molecules per antibody

• Purity: Typically >95% for research-grade antibodies

• Formulation: Buffer components (e.g., PBS, pH, preservatives)

• Storage conditions: Usually -20°C with recommendations to avoid freeze-thaw cycles

What are the primary applications for biotin-conjugated WDR83 antibodies?

Biotin-conjugated WDR83 antibodies can be utilized in various experimental approaches:

• ELISA: Both sandwich and capture formats for the direct binding of antigen

• Western blotting: For detecting WDR83 in protein lysates

• Immunohistochemistry (IHC): For tissue localization of WDR83

• Immunofluorescence (IF): For cellular localization studies

• Flow cytometry (FC): For analyzing WDR83 expression in cell populations

• Multiplex assays: For simultaneous detection of multiple targets

• Proximity-dependent biotinylation: For studying protein-protein interactions

How does the biotin-streptavidin system enhance detection sensitivity in WDR83 studies?

The biotin-streptavidin system significantly enhances detection sensitivity through several mechanisms:

• Signal amplification: Each streptavidin molecule can bind four biotin molecules, allowing for enhanced signal through multiple secondary detection reagents binding to each primary antibody

• High affinity binding: The extraordinary affinity (Kd ~10^-15 M) ensures stable and specific detection even in stringent washing conditions

• Versatility in detection methods: Compatible with various detection systems:

  • Enzyme-conjugated streptavidin (HRP or AP) for colorimetric or chemiluminescent detection

  • Fluorophore-conjugated streptavidin for fluorescence microscopy or flow cytometry

  • Gold-conjugated streptavidin for electron microscopy

  • Quantum dot-conjugated streptavidin for photostable fluorescence detection

• Improved sensitivity: The system can detect WDR83 at concentrations as low as 50 ng/mL in some assay formats

What is the recommended protocol for using biotin-conjugated WDR83 antibody in Western blotting?

Standard Western Blotting Protocol for Biotin-Conjugated WDR83 Antibody:

• Sample preparation:

  • Prepare cell/tissue lysates in appropriate lysis buffer

  • Determine protein concentration (e.g., BCA assay)

  • Mix samples with loading buffer and denature at 95°C for 5 minutes

• Gel electrophoresis:

  • Load 10-30 μg protein per lane on SDS-PAGE gel

  • Note: WDR83 may appear at approximately 12 kDa despite calculated MW of ~34 kDa

• Transfer:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Blocking:

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

  • Note: Avoid using biotin-containing blocking reagents

• Primary antibody incubation:

  • Dilute biotin-conjugated WDR83 antibody (optimal dilutions should be determined by end user)

  • Incubate membrane overnight at 4°C

• Washing:

  • Wash membrane 3x for 5 minutes each with TBST

• Detection:

  • Incubate with streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature

  • Wash 3x for 5 minutes each with TBST

  • Develop using ECL substrate and image membrane

• Controls:

  • Include positive control (known WDR83-expressing sample)

  • Include negative control (samples without WDR83 expression)

  • Consider including endogenously biotinylated protein control to identify potential background

How can metabolic biotinylation efficiency be enhanced when studying WDR83 interactions?

When investigating WDR83 protein interactions using proximity-dependent biotinylation methods like BioID, researchers can enhance biotinylation efficiency through several strategies:

• Co-expression of bacterial biotin ligase: Overexpression of bacterial biotin ligase (BirA) in the same cellular compartment as the fusion protein can enhance biotinylation by more than 10-fold

• Optimization of biotin ligase localization: Direct the biotin ligase to the appropriate cellular compartment (cytoplasm, secretory pathway, or ER) for maximum efficiency:

  • Cytoplasmic proteins: use hBirA

  • Secreted or membrane proteins: use sshBirA

  • ER-resident proteins: use sshBirA-KDEL

• Use of enhanced biotin ligase variants: Employ more efficient enzymes like TurboID or ultraID, which offer:

  • Faster labeling kinetics (10 minutes vs. hours for traditional BioID)

  • Smaller size (ultraID is the smallest available biotin ligase for PDB)

  • Higher labeling efficiency at physiological expression levels

• Optimization of biotin concentration and exposure time: Adjust exogenous biotin concentration and labeling duration to balance signal-to-noise ratio and prevent saturation

• Cell type consideration: Account for varying efficiencies of metabolic biotinylation in different cell types due to differences in endogenous biotin ligase expression levels

What approaches can resolve inconsistent results when using biotin-conjugated WDR83 antibodies in multiplex assays?

Inconsistent results in multiplex assays using biotin-conjugated WDR83 antibodies may arise from various factors. To resolve these issues:

• Characterize biotin incorporation ratio: Determine the biotin-to-antibody ratio using biotin quantification assays. An optimal range is typically 3-8 biotin molecules per antibody; over-biotinylation can compromise antigen binding

• Perform functional assessments: Compare raw response values (median fluorescence intensity, molecules of equivalent soluble fluorochrome) between different lots or preparations

• Prepare dilution series: Create a standard curve with serially diluted antibody to identify the linear detection range and optimal working concentration

• Control for endogenous biotinylated proteins: Use appropriate controls to identify and subtract signal from endogenously biotinylated proteins, which may cause background issues

• Optimize detection reagent: Test different streptavidin conjugates (HRP, AP, fluorophores) to identify the optimal signal-to-noise ratio for your specific multiplex system

How can researchers validate the specificity of WDR83 detection in complex tissue samples using biotin-conjugated antibodies?

Validating specificity of WDR83 detection in complex samples requires multiple complementary approaches:

• Multiple detection methods: Confirm findings using independent techniques:

  • Western blot to verify molecular weight (note the discrepancy between calculated MW ~34 kDa and observed ~12 kDa)

  • Immunoprecipitation followed by mass spectrometry

  • Orthogonal detection methods (e.g., RNA-seq, in situ hybridization)

• Pre-adsorption controls: Perform parallel experiments with:

  • Regular biotin-conjugated WDR83 antibody

  • Antibody pre-incubated with purified WDR83 antigen (should show reduced signal)

  • Isotype-matched biotinylated control antibody (should show no specific signal)

• Genetic validation: Use samples with manipulated WDR83 expression:

  • WDR83 knockout or knockdown tissues/cells (should show reduced/absent signal)

  • WDR83 overexpression systems (should show enhanced signal)

• Cross-reactivity assessment: Test antibody against closely related WD-repeat domain proteins to ensure specificity

• Spatial distribution analysis: Compare observed cellular/subcellular localization with known or predicted WDR83 expression patterns

• Mass spectrometry verification: For proximity labeling studies, use direct detection of biotin-containing tags (DiDBiT) and Neutravidin protein enrichment strategies to confirm the identity of biotinylated proteins

What are the common pitfalls when using biotin-conjugated antibodies in WDR83 research and how can they be addressed?

Common Problems and Solutions:

• High background signal:

  • Cause: Endogenous biotinylated proteins or excessive biotinylation of antibody

  • Solution: Use streptavidin blocking reagents, include avidin/biotin blocking steps, optimize antibody dilution, or incorporate biotin-free blocking buffers

• Weak or no signal:

  • Cause: Insufficient biotinylation, degraded antibody, or low target abundance

  • Solution: Verify biotin incorporation ratio, use fresh antibody aliquots, increase sample concentration, or employ signal amplification strategies

• Non-specific bands in Western blots:

  • Cause: Cross-reactivity or detection of endogenous biotinylated proteins

  • Solution: Increase antibody dilution, optimize washing steps, pre-adsorb antibody against problematic epitopes, or include competitor proteins in blocking solution

• Variable results between experiments:

  • Cause: Lot-to-lot variation or inconsistent handling

  • Solution: Characterize new lots before use, standardize protocols, create internal reference standards, or prepare larger single-lot stocks for long-term studies

• Reduced antibody efficacy after storage:

  • Cause: Repeated freeze-thaw cycles or improper storage

  • Solution: Aliquot antibody upon receipt, store at recommended temperature (-20°C), avoid freeze-thaw cycles, and add carrier protein for diluted antibodies

How can researchers optimize buffer conditions for maximum sensitivity when using biotin-conjugated WDR83 antibodies?

Optimizing buffer conditions is critical for maximizing sensitivity and specificity:

• Primary antibody diluent optimization:

  • Base buffer: Use 10-50 mM amine-free buffer (HEPES, MES, MOPS, phosphate) at pH 6.5-8.5

  • Avoid: Buffers containing nucleophilic components (primary amines), thiols (Thiomersal/Thimerosal), Merthiolate, Glycine or Proclin, which may interfere with biotin-streptavidin interactions

  • Compatible additives: Azide (0.02-0.1%), EDTA, and common non-buffering salts and sugars have minimal effect on conjugation efficiency

  • Protein additives: 1-5% BSA or casein (biotin-free) can reduce background

• Washing buffer optimization:

  • Increase washing buffer stringency (0.1-0.5% Tween-20) to reduce non-specific binding

  • Consider adding low concentrations of salt (150-500 mM NaCl) to reduce ionic interactions

  • Maintain consistent pH (typically 7.2-7.6) for optimal streptavidin-biotin binding

• Detection reagent optimization:

  • Streptavidin conjugate selection: Compare streptavidin-HRP, streptavidin-AP, or fluorophore-conjugated streptavidin for optimal signal-to-noise ratio

  • Incubation conditions: Optimize temperature (room temperature vs. 4°C) and time (30 min - overnight)

  • Consider neutravidin: For applications with high background, neutravidin may provide reduced non-specific binding compared to streptavidin or avidin

What are the critical factors for successful biotinylation of WDR83 antibodies in a laboratory setting?

For researchers preparing their own biotin-conjugated WDR83 antibodies, several critical factors should be considered:

• Antibody preparation:

  • Use highly purified antibody (>95% purity)

  • Ensure antibody is in appropriate buffer (10-50 mM amine-free buffer, pH 6.5-8.5)

  • Optimal concentration range: 1-2.5 mg/ml

  • Remove any nucleophilic components or thiols from the buffer

• Biotinylation reagent selection:

  • Choose appropriate biotin derivative:

    • NHS-biotin: standard reagent for primary amine labeling

    • Sulfo-NHS-LC-biotin: water-soluble with extended spacer arm

    • Maleimide-PEG-biotin: for thiol-specific labeling

  • Consider spacer length: longer spacers (like in Biotin-SP) improve accessibility and sensitivity

• Reaction conditions optimization:

  • Biotin-to-antibody molar ratio: typically 10:1 to 20:1 for optimal labeling

  • Reaction time and temperature: usually 30-60 minutes at room temperature

  • Quenching: terminate reaction with primary amine-containing buffer

• Purification of conjugate:

  • Remove unreacted biotin using desalting columns

  • Equilibrate columns with appropriate storage buffer (e.g., MSD® Conjugate Storage Buffer)

  • Verify purification efficiency

• Conjugate characterization:

  • Determine total protein concentration

  • Measure biotin incorporation ratio:

    • Optimal range: 3-8 biotin molecules per antibody

    • Over-biotinylation (>10 biotin/antibody) may compromise antigen binding

  • Test functional activity in the intended application

• Storage considerations:

  • Aliquot to avoid freeze-thaw cycles

  • Store at -20°C

  • Add carrier protein (BSA) for long-term stability

  • Consider adding preservatives (0.02-0.1% sodium azide) for microbial control

How is proximity-dependent biotinylation being utilized to study WDR83 protein interactions?

Proximity-dependent biotinylation (PDB) represents an advanced approach for studying WDR83 protein interactions in living cells:

• BioID methodology application:

  • Fusion of an abortive biotin ligase (BirA R118G) to WDR83

  • Expression in cells with exogenous biotin supplementation

  • Biotinylation of proteins within approximately 10 nm radius

  • Affinity purification of biotinylated proteins using streptavidin

  • Mass spectrometry identification of interaction partners

• Advanced enzyme options:

  • TurboID: Enhanced activity allowing 10-minute labeling versus hours for traditional BioID

  • miniTurbo: Smaller size with improved labeling speed

  • ultraID: The smallest and most efficient biotin ligase available for PDB, offering high temporal resolution

• Cell type-specific approaches:

  • Transgenic expression of TurboID under cell-type-specific promoters

  • Adenoviral delivery for regional expression in specific tissues

  • Enables mapping of cell type-specific interactomes in complex tissues

• Advantages for WDR83 research:

  • Captures transient or weak interactions difficult to detect by co-IP

  • Works under physiological conditions in living cells

  • Can identify compartment-specific interaction networks

  • Compatible with various cell types and in vivo applications

What innovations in biotin-conjugated antibody technology are enabling new approaches to WDR83 research?

Recent technological advances are expanding the capabilities of biotin-conjugated antibodies in WDR83 research:

• Nanobody-based proximity biotinylation:

  • Development of nanobody-based PDB systems that can be used to define the interactome of any GFP-tagged protein

  • Application to GFP-tagged WDR83 enables identification of interaction partners without creating new fusion constructs

  • Higher spatial resolution due to smaller size of nanobodies compared to conventional antibodies

• Biotin conjugation with cleavable linkers:

  • Incorporation of stimuli-responsive linkers between biotin and antibody

  • Enables controlled release of captured proteins under specific conditions

  • Useful for studying dynamic protein complexes or sequential interaction events

• Multimodal labeling strategies:

  • Dual-labeled antibodies combining biotin with fluorophores or other detection modalities

  • Enables simultaneous visualization and affinity purification

  • Applications in correlative microscopy approaches combining light and electron microscopy

• Quantitative biotinylation approaches:

  • Development of MS/MS methods specifically for biotin-labeled proteins:

    • Direct detection of biotin-containing tags (DiDBiT)

    • Enhanced neutravidin protein enrichment strategies

  • Enables quantitative comparison of WDR83 interactomes under different conditions

What does current research reveal about WDR83's functional roles based on studies using biotin-conjugated antibodies?

While the search results don't provide specific findings about WDR83's functional roles using biotin-conjugated antibodies, general principles can be applied to interpret research in this area:

• Protein complex identification:

  • Biotin-conjugated WDR83 antibodies combined with streptavidin pull-down can identify stable protein complexes containing WDR83

  • Mass spectrometry analysis of these complexes may reveal functional protein networks

• Subcellular localization:

  • Immunohistochemistry or immunofluorescence using biotin-conjugated WDR83 antibodies can reveal the protein's distribution in cells and tissues

  • WDR83's localization pattern may provide clues about its function in different cellular compartments

• Expression pattern analysis:

  • Studies of WDR83 expression across different tissues, developmental stages, or disease states using biotin-conjugated antibodies can highlight conditions where this protein plays important roles

  • Quantitative approaches like ELISA can measure changes in WDR83 levels under various conditions

• Functional studies:

  • Biotin-conjugated antibodies can be used to block WDR83 function in certain experimental settings

  • The resulting phenotypic changes may reveal the protein's physiological roles

• Interactome mapping:

  • Proximity labeling approaches fusing biotin ligases to WDR83 can identify its protein interaction network

  • These interactomes may connect WDR83 to specific cellular pathways and functions

Future research employing these approaches will likely continue to elucidate WDR83's roles in normal physiology and disease states.