WDR83 Antibody, FITC conjugated

What is WDR83 and what cellular functions does it regulate?

WDR83 (also known as MORG1, MAPK organizer 1, or WD repeat domain-containing protein 83) functions as a molecular scaffold protein for various multimeric protein complexes. It serves as a module in the assembly of a multicomponent scaffold for the ERK pathway, connecting ERK responses to specific agonists. At low concentrations, WDR83 enhances ERK activation, while high concentrations inhibit ERK activation. Additionally, WDR83 is involved in hypoxia response by negatively regulating HIF1A/HIF-1-alpha through its interaction with EGLN3/PHD3. It may promote degradation of HIF1A and may also participate in pre-mRNA splicing processes .

What are the technical specifications of the WDR83 Antibody, FITC conjugate?

The WDR83 Antibody, FITC conjugate is a polyclonal antibody raised in rabbits against a recombinant WD repeat domain-containing protein 83 protein fragment (amino acids 64-315). It has been purified using Protein G (>95% purity) and conjugated with fluorescein isothiocyanate (FITC). The antibody is supplied in liquid form in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. The FITC fluorophore typically has excitation/emission maxima wavelengths of 494 nm and 520 nm, respectively, which is consistent with standard FITC-conjugated reagents .

How should WDR83 Antibody, FITC conjugate be stored to maintain optimal activity?

For proper storage of the WDR83 Antibody, FITC conjugate, it is recommended to store the reagent at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can deteriorate antibody quality and reduce fluorescence intensity. As with other FITC-conjugated antibodies, it's crucial to protect the reagent from light exposure, as FITC is photosensitive and can photobleach, resulting in reduced signal intensity. When stored properly, FITC-conjugated antibodies typically maintain stability for at least one year after shipment .

How can WDR83 Antibody, FITC conjugate be used to study the role of WDR83 in ERK pathway regulation?

To study WDR83's role in ERK pathway regulation, researchers can employ the WDR83 Antibody, FITC conjugate in multiparameter flow cytometry experiments combined with phospho-specific antibodies against ERK1/2. This approach allows for simultaneous detection of WDR83 expression levels and ERK activation status at the single-cell level.

Methodology:

• Treat cells with varying concentrations of pathway agonists (e.g., growth factors)

• Fix and permeabilize cells using an appropriate protocol compatible with both surface and intracellular staining

• Stain with WDR83 Antibody, FITC conjugate and a complementary fluorochrome-conjugated phospho-ERK1/2 antibody

• Analyze by flow cytometry, comparing WDR83 expression levels with ERK phosphorylation status

This experimental design can verify the concentration-dependent regulation of ERK activation by WDR83, where low WDR83 levels enhance ERK activation while high levels inhibit it. Correlation analysis between WDR83 fluorescence intensity and phospho-ERK signals can provide quantitative insights into this relationship .

What approaches can be used to investigate WDR83's interaction with the hypoxia response pathway using the FITC-conjugated antibody?

Investigating WDR83's role in hypoxia response pathways requires experimental designs that capture its interaction with HIF1A and EGLN3/PHD3. The FITC-conjugated WDR83 antibody can be employed in several advanced approaches:

• Co-localization studies:

  • Culture cells under normoxic and hypoxic conditions

  • Fix and permeabilize cells

  • Perform co-staining with WDR83 Antibody, FITC conjugate and fluorescently-labeled antibodies against HIF1A and EGLN3 (using different fluorophores)

  • Analyze using confocal microscopy or imaging flow cytometry

  • Quantify co-localization coefficients to measure spatial associations

• Hypoxia response experiments:

  • Expose cells to hypoxic conditions at different timepoints

  • Measure WDR83 expression levels using flow cytometry with the FITC-conjugated antibody

  • Simultaneously assess HIF1A stability and target gene expression

  • Compare results between WDR83 knockdown and control cells

This comprehensive approach can reveal the dynamic relationship between WDR83 expression and hypoxia response regulation, particularly focusing on its negative regulatory effect on HIF1A stability .

How can protein complex formation involving WDR83 be analyzed using FITC-conjugated WDR83 antibody?

As WDR83 functions as a scaffold protein for multimeric protein complexes, analyzing these interactions is crucial. The FITC-conjugated WDR83 antibody can be utilized in several sophisticated experimental approaches:

• Proximity ligation assay (PLA) combined with fluorescence microscopy:

  • Fix and permeabilize cells

  • Incubate with WDR83 Antibody, FITC conjugate and unconjugated antibodies against suspected interaction partners

  • Perform PLA according to manufacturer's protocol

  • Visualize interaction signals alongside WDR83-FITC localization

  • Quantify interaction frequency in different cellular compartments

• Flow cytometry-based protein interaction analysis:

  • Immunoprecipitate protein complexes using anti-WDR83 antibody

  • Label precipitated complexes with fluorescently-tagged antibodies against interaction partners

  • Analyze using flow cytometry to detect co-precipitated proteins

  • Compare complex formation under different cellular conditions

These methodologies enable researchers to identify and characterize the components of WDR83-containing protein complexes and how they change under different signaling conditions or cellular stresses .

What are the optimal conditions for using WDR83 Antibody, FITC conjugate in flow cytometry experiments?

For optimal results in flow cytometry experiments using WDR83 Antibody, FITC conjugate, consider the following methodological parameters:

• Sample preparation:

  • For cell suspensions: Use approximately 4 μl of antibody per 10^6 cells in 100 μl buffer

  • For adherent cells: Ensure complete dissociation into single-cell suspensions before staining

  • Maintain cells at 4°C during staining to prevent internalization

• Buffer composition:

  • Use PBS with 1-2% protein (BSA or FBS) and 0.1% sodium azide

  • Adjust pH to 7.2-7.4 for optimal antibody binding

  • Include 2mM EDTA when working with cells that tend to aggregate

• Staining protocol:

  • For surface staining: Incubate cells with antibody for 20-30 minutes at 4°C in the dark

  • For intracellular staining: Fix cells with 2-4% paraformaldehyde, then permeabilize with 0.1% saponin or 0.3% Triton X-100 before antibody incubation

  • Include a fixable viability dye to exclude dead cells from analysis

• Instrument settings:

  • Excite with a 488 nm laser

  • Collect emission using a 520/30 nm bandpass filter

  • Perform proper compensation if using multiple fluorochromes

• Controls:

  • Include unstained cells, isotype control, and single-stained controls for compensation

  • Consider fluorescence-minus-one (FMO) controls for accurate gating

These parameters should be optimized for each specific cell type and experimental condition .

What is the molecular basis of FITC conjugation to the WDR83 antibody and how does this affect antibody performance?

The molecular process of FITC conjugation to WDR83 antibody involves the reaction between the isothiocyanate group of FITC and primary amines (mainly lysine residues) on the antibody protein. This results in the formation of stable thiourea bonds. The conjugation process and its impact on antibody performance can be understood through these key aspects:

• Chemistry of conjugation:

  • Optimal conjugation occurs at pH 9.5, where lysine side chains are deprotonated

  • The reaction typically requires 30-60 minutes at room temperature with an initial protein concentration of 25 mg/ml

  • The degree of labeling, expressed as the fluorescein/protein (F/P) ratio, is critical for antibody performance

• Effects on antibody structure and function:

  • Over-labeling (high F/P ratio) can interfere with antigen binding by modifying lysines in or near the antigen-binding site

  • Excessive FITC conjugation may alter antibody conformation and increase hydrophobicity

  • The optimal F/P ratio typically ranges from 3-8 molecules of FITC per antibody molecule

• Performance characteristics:

  • Properly conjugated antibodies maintain specificity while gaining fluorescence detection capability

  • Signal-to-noise ratio depends on the F/P ratio, with higher ratios providing brighter signals but potentially higher background

  • The process of conjugation may slightly reduce antibody affinity in some cases

• Purification considerations:

  • After conjugation, separation of optimally labeled antibodies from under- and over-labeled proteins is achieved through gradient DEAE Sephadex chromatography

  • This purification step is crucial for ensuring consistent performance in experiments

Understanding these molecular aspects helps researchers interpret results and troubleshoot issues related to antibody performance in their experimental systems .

How can the specific binding of WDR83 Antibody, FITC conjugate be validated in experimental systems?

Validating the specific binding of WDR83 Antibody, FITC conjugate is essential for ensuring reliable experimental results. A comprehensive validation approach should include:

• Positive and negative control samples:

  • Positive controls: Cell lines with confirmed WDR83 expression (based on literature or genomic databases)

  • Negative controls: Cell lines with low/no WDR83 expression or WDR83 knockout models

  • Compare staining patterns between these controls using flow cytometry or microscopy

• Competitive binding assays:

  • Pre-incubate cells with excess unconjugated anti-WDR83 antibody

  • Follow with FITC-conjugated WDR83 antibody at standard concentration

  • A significant reduction in fluorescence signal indicates specific binding

• Recombinant protein blocking:

  • Pre-incubate the FITC-conjugated antibody with purified recombinant WDR83 protein

  • Apply this mixture to cells and compare with unblocked antibody staining

  • Specific binding is indicated by reduced staining after blocking

• Genetic validation:

  • Perform siRNA/shRNA knockdown of WDR83

  • Compare antibody staining in knockdown vs. control cells

  • Reduced signal in knockdown cells confirms specificity

• Western blot correlation:

  • Sort cell populations based on WDR83-FITC staining intensity

  • Perform Western blot analysis on sorted populations

  • Correlation between fluorescence intensity and protein band intensity validates specificity

These validation steps ensure that experimental observations reflect true WDR83 biology rather than artifacts or non-specific binding .

How can researchers address weak signal issues when using WDR83 Antibody, FITC conjugate?

When encountering weak signal problems with WDR83 Antibody, FITC conjugate, researchers should systematically investigate potential causes and apply appropriate solutions:

• Antibody-related factors:

  • Check antibody storage conditions (exposure to light, temperature fluctuations)

  • Verify antibody expiration date and lot-to-lot variations

  • Solution: Use fresh aliquots and avoid repeated freeze-thaw cycles

• Sample preparation issues:

  • Insufficient permeabilization for intracellular targets

  • Inadequate fixation leading to antigen loss

  • Solution: Optimize fixation and permeabilization protocols; try different detergents or concentrations

• Technical parameters:

  • Suboptimal antibody concentration

  • Insufficient incubation time

  • Solution: Perform titration experiments to determine optimal concentration; extend incubation time to 45-60 minutes

• Target protein expression:

  • Low WDR83 expression in the studied cell type

  • Expression changes under experimental conditions

  • Solution: Use positive control cells with known WDR83 expression; consider signal amplification methods

• Instrument settings:

  • Improper voltage settings on flow cytometer

  • Incorrect filter selection

  • Solution: Optimize PMT voltages; ensure proper filters for FITC detection (520/30 nm bandpass)

• Fluorophore considerations:

  • FITC photobleaching during sample preparation or analysis

  • pH sensitivity of FITC (fluorescence decreases at lower pH)

  • Solution: Minimize light exposure; maintain buffer pH between 7.2-8.0

A systematic approach to these factors will help identify and address the specific cause of weak signal issues .

What strategies can be employed to distinguish between specific and non-specific signals when analyzing WDR83 localization?

Distinguishing between specific and non-specific signals is crucial for accurate interpretation of WDR83 localization studies using FITC-conjugated antibodies. Researchers should implement these methodological strategies:

• Comprehensive controls:

  • Isotype controls: Match the host species, isotype, and FITC labeling density

  • Blocking controls: Pre-incubate with recombinant WDR83 protein

  • Secondary-only controls (if using indirect methods)

  • Autofluorescence controls: Unstained samples to establish baseline

  • Genetic controls: WDR83 knockdown or knockout samples

• Multi-parameter confirmation:

  • Co-staining with antibodies against known WDR83-interacting proteins

  • Correlation of localization patterns with expected biological contexts

  • Comparison of staining patterns across multiple cell types with different WDR83 expression levels

• Signal characterization:

  • Evaluate signal-to-noise ratio across different cellular compartments

  • Compare staining intensity with Western blot quantification

  • Analyze pixel intensity distributions in microscopy images

• Manipulation experiments:

  • Induce known biological changes that affect WDR83 (e.g., hypoxia)

  • Observe expected relocalization or expression changes

  • Compare with published literature on WDR83 behavior

• Technical approaches:

  • Spectral unmixing to separate FITC signal from autofluorescence

  • Image processing algorithms to enhance specific signals

  • Super-resolution microscopy for detailed localization analysis

By implementing these strategies, researchers can confidently distinguish between specific WDR83 localization and background or non-specific fluorescence, leading to more reliable data interpretation .

How can researchers quantitatively analyze WDR83 expression levels using FITC-conjugated antibodies in heterogeneous cell populations?

Quantitative analysis of WDR83 expression in heterogeneous populations requires sophisticated approaches that account for cellular diversity while maintaining analytical rigor:

• Multiparameter flow cytometry:

  • Design a panel with lineage markers to identify cell subsets

  • Include WDR83 Antibody, FITC conjugate and relevant functional markers

  • Calculate median fluorescence intensity (MFI) for WDR83 in each defined population

  • Apply standardization using calibration beads to convert to molecules of equivalent soluble fluorochrome (MESF)

• Statistical analysis approaches:

  • Population comparison: Use appropriate statistical tests (t-test, ANOVA) to compare WDR83 expression between defined populations

  • Correlation analysis: Assess relationships between WDR83 levels and other parameters

  • Distribution analysis: Examine expression histograms for evidence of distinct subpopulations

• Imaging cytometry methods:

  • Combine flow cytometry with imaging capabilities

  • Analyze subcellular localization alongside expression levels

  • Correlate morphological features with WDR83 expression

• Single-cell analysis:

  • Index sorting followed by single-cell transcriptomics or proteomics

  • Correlation of WDR83 protein levels (by FITC fluorescence) with mRNA expression

  • Identification of co-expression patterns with other genes/proteins

• Quantification standards:

  • Include quantitative calibration standards in each experiment

  • Calculate absolute protein quantities using standard curves

  • Ensure batch-to-batch consistency with reference samples

These approaches enable robust quantitative analysis of WDR83 expression across complex cell populations, revealing biological patterns that might be obscured in bulk analyses .

How can WDR83 Antibody, FITC conjugate be integrated into high-content screening workflows?

Integrating WDR83 Antibody, FITC conjugate into high-content screening (HCS) workflows offers powerful approaches for drug discovery and functional genomics research related to ERK pathway modulation and hypoxia response:

• Automated imaging platform setup:

  • Optimize cell seeding density in 96/384-well plates for consistent imaging

  • Establish fixed exposure settings for FITC channel to enable quantitative comparisons

  • Develop automated image acquisition protocols that capture sufficient cells per well (>500)

  • Create analysis pipelines that segment cells and quantify WDR83-FITC intensity and localization

• Multiplexed assay design:

  • Combine WDR83-FITC staining with nuclear counterstains (e.g., DAPI)

  • Include additional markers for cell health, signaling pathway activation, or phenotypic outcomes

  • Design assays that correlate WDR83 expression with functional endpoints

• Screening methodology:

  • Primary screen: Measure changes in WDR83 expression or localization in response to compound libraries or genetic perturbations

  • Secondary assays: Validate hits with orthogonal measurements of pathway activity

  • Dose-response studies: Determine concentration-dependent effects on WDR83 biology

• Data analysis framework:

  • Establish normal ranges and thresholds for significant changes

  • Apply machine learning algorithms to identify complex phenotypes

  • Integrate with other -omics datasets for comprehensive interpretation

This approach enables systematic investigation of compounds or genetic factors that modulate WDR83 expression, localization, or function in high-throughput formats, accelerating discovery in cancer research, cardiovascular disease, and hypoxia-related pathologies .

What considerations are important when designing experiments to study WDR83 dynamics in live cell imaging?

While the FITC-conjugated WDR83 antibody is primarily designed for fixed cell applications, researchers interested in studying WDR83 dynamics in live cells should consider these alternative approaches and important considerations:

• Alternative labeling strategies:

  • Generate fluorescent protein fusions (e.g., WDR83-GFP) through transfection or CRISPR knock-in

  • Use cell-permeable fluorogenic antibody fragments or nanobodies

  • Consider photoactivatable or photoconvertible fluorescent proteins for pulse-chase experiments

• Physiological relevance:

  • Validate that tagged WDR83 maintains normal localization and function

  • Express at or near endogenous levels to avoid artifacts

  • Compare dynamics with fixed-cell antibody staining as a reference

• Imaging parameters:

  • Minimize phototoxicity through reduced exposure times and light intensity

  • Use sensitive cameras and objectives with high numerical aperture

  • Balance temporal resolution with signal-to-noise considerations

  • Consider resonant scanning confocal or spinning disk systems for faster acquisition

• Environmental controls:

  • Maintain physiological temperature, CO2, and humidity

  • Design experiments to capture both short-term (seconds to minutes) and long-term (hours) dynamics

  • Include appropriate controls for photobleaching and phototoxicity

• Analysis approaches:

  • Track individual WDR83-containing structures over time

  • Measure protein turnover through FRAP (Fluorescence Recovery After Photobleaching)

  • Quantify co-localization dynamics with other labeled proteins

  • Apply computational methods to extract motion parameters and interaction kinetics

These considerations help researchers design live-cell imaging experiments that reveal the dynamic properties of WDR83 in physiologically relevant contexts, complementing fixed-cell studies using the FITC-conjugated antibody .

How does the performance of polyclonal WDR83 Antibody, FITC conjugate compare with monoclonal alternatives for specific research applications?

Understanding the comparative advantages and limitations of polyclonal versus monoclonal WDR83 antibodies is essential for selecting the appropriate reagent for specific research applications:

For research focusing on WDR83's role in ERK pathway regulation or hypoxia response, these considerations guide antibody selection:

• For detecting total WDR83 expression levels:

  • Polyclonal WDR83 Antibody, FITC offers higher sensitivity by recognizing multiple epitopes

  • Beneficial when protein expression is low or variable

• For specific localization studies:

  • Monoclonal antibodies provide more consistent staining patterns

  • Better for co-localization studies with other proteins

• For detecting specific WDR83 protein interactions:

  • Epitope-specific monoclonal antibodies prevent interference with interaction sites

  • Critical when studying specific protein-protein interfaces

When selecting between polyclonal and monoclonal WDR83 antibodies, researchers should consider their specific experimental requirements, balancing sensitivity, specificity, and reproducibility needs .

What emerging technologies could enhance the utility of WDR83 Antibody, FITC conjugate in multi-omics research approaches?

Integrating WDR83 Antibody, FITC conjugate with emerging multi-omics technologies opens new frontiers for understanding WDR83 biology in complex cellular contexts:

• Spatial proteomics approaches:

  • Imaging mass cytometry: Combine WDR83-FITC antibody with metal-tagged antibodies for simultaneous detection of >40 proteins

  • CODEX multiplexed imaging: Iterative antibody staining to correlate WDR83 with extensive protein networks

  • Application: Map WDR83 distribution relative to signaling networks in tissue microenvironments

• Single-cell multi-omics integration:

  • CITE-seq: Couple WDR83 protein detection with transcriptome analysis

  • Flow-sorting WDR83-FITC labeled populations for downstream proteomics or metabolomics

  • Application: Correlate WDR83 protein levels with global gene expression patterns

• Proximity labeling technologies:

  • Conjugate proximity labeling enzymes (BioID, APEX) to anti-WDR83 antibodies

  • Identify proteins in close proximity to WDR83 under different conditions

  • Application: Discover context-specific WDR83 interaction networks

• Advanced microscopy techniques:

  • Super-resolution microscopy: Combine with FITC-conjugated WDR83 antibody for nanoscale localization

  • Lattice light-sheet microscopy: Capture dynamic WDR83 interactions with reduced phototoxicity

  • Application: Resolve subcellular distribution and trafficking of WDR83-containing complexes

• Functional genomics integration:

  • CRISPR screens with WDR83-FITC readouts to identify regulators

  • Correlate genetic perturbations with changes in WDR83 expression or localization

  • Application: Systematic identification of genes affecting WDR83 function

These emerging technologies will advance understanding of WDR83's role in coordinating cellular responses to stimuli, particularly in the context of ERK pathway regulation and hypoxia response mechanisms .