WDR35 Antibody, Biotin conjugated

What is the target specificity of commercially available WDR35 biotin-conjugated antibodies?

Most commercial WDR35 biotin-conjugated antibodies target specific amino acid sequences within the protein. The majority of antibodies are developed against regions including AA 954-1181 or similar segments of the human WDR35 protein . When selecting an antibody for your research, it's crucial to verify that the epitope region corresponds to the domain of interest in your experimental design. The specificity can be confirmed through validation data showing single band detection in Western blots or specific cellular localization in immunofluorescence studies. Cross-reactivity data shows that while most are designed for human samples, some antibodies also react with canine samples and other species .

What detection methods are compatible with biotin-conjugated WDR35 antibodies?

Biotin-conjugated WDR35 antibodies are particularly versatile for multiple detection platforms including:

• ELISA: The biotin conjugation enables highly sensitive sandwich ELISA applications with detection limits as low as 0.062 ng/ml .

• Immunohistochemistry: Following streptavidin-HRP incubation, DAB visualization allows for tissue-specific localization studies .

• Immunofluorescence microscopy: When combined with fluorophore-conjugated streptavidin, these antibodies enable high-resolution imaging of ciliary structures .

The biotin-streptavidin interaction's high affinity (Kd ≈ 10^-15 M) provides significant signal amplification, which is particularly valuable when studying low-abundance proteins like WDR35 in ciliary structures .

How should biotin-conjugated WDR35 antibodies be stored to maintain optimal activity?

For maximum stability, biotin-conjugated WDR35 antibodies should be stored at -20°C in aliquots to avoid repeated freeze-thaw cycles. Most manufacturers provide these antibodies in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibodies typically remain stable for one year after shipment. It's important to note that larger volume formats (>20μl) may not contain BSA, while smaller formats (20μl) often contain 0.1% BSA for additional stability .

How can biotin-conjugated WDR35 antibodies be used to investigate WDR35's role in ciliary transport mechanisms?

Recent studies have established WDR35 as a key component of a COPI-like complex that delivers proteins to growing cilia . To investigate this function, researchers can employ correlative light and electron microscopy (CLEM) using biotin-conjugated WDR35 antibodies. This methodology allows for:

• Visualization of WDR35-positive vesicles at the ciliary base

• Identification of electron-dense coated vesicles in TEM images

• Confirmation of WDR35's involvement in vesicle formation through rescue experiments

For optimal results, the following protocol has proven effective:

• Fix cells using 4% PFA followed by careful permeabilization

• Block with 4% BSA in PBS-T to reduce background

• Apply biotin-conjugated WDR35 antibody (1:200-1:500 dilution)

• Visualize using streptavidin-conjugated gold particles (15nm) for EM studies

This approach has successfully demonstrated that WDR35 supports the assembly of a novel coat on vesicles destined for ciliary membrane cargo delivery .

Can biotin-conjugated WDR35 antibodies be used to investigate the relationship between WDR35 expression and oxidative stress?

Studies have indicated that reactive oxygen species (ROS) play a significant role in the regulation of WDR35 expression. Specifically, research has shown that the radical scavenger edaravone significantly attenuates domoic acid (DA)-induced upregulation of WDR35 in rat hippocampus .

To investigate this relationship, biotin-conjugated WDR35 antibodies can be employed in a multifaceted approach:

• Treat cellular models with oxidative stress inducers (H₂O₂, rotenone, etc.)

• Apply ROS scavengers to experimental groups

• Quantify WDR35 expression levels using biotin-conjugated antibodies in ELISA format

• Visualize cellular localization changes using streptavidin-fluorophore detection

This methodological approach allows researchers to determine whether oxidative stress induces not only changes in WDR35 expression levels but also potential alterations in subcellular distribution, particularly in relation to ciliary structures .

What approaches can resolve contradictory data when using biotin-conjugated WDR35 antibodies in multi-omics studies?

When integrating antibody-based detection with genomic or proteomic datasets, researchers occasionally encounter discrepancies. To resolve such contradictions:

• Epitope availability verification: Perform parallel experiments with antibodies targeting different WDR35 epitopes to rule out conformational masking issues.

• Cross-validation approach: Implement the following strategy:

• Site-specific controls: For ciliary localization studies, co-staining with established ciliary markers (ARL13B, acetylated tubulin) and basal body markers (γ-tubulin) to confirm spatial relationships .

What is the optimal sandwich ELISA protocol for detecting WDR35 using biotin-conjugated antibodies?

For maximum sensitivity and specificity when quantifying WDR35 in biological samples, the following optimized sandwich ELISA protocol is recommended:

• Coat microwell plates with capture antibody (anti-WDR35, unconjugated) at 2μg/ml in carbonate buffer (pH 9.6), incubate overnight at 4°C

• Wash and block wells with 4% BSA in PBS-T for 1 hour at room temperature

• Add samples and standards (range: 0.156-10 ng/ml), incubate for 2 hours at room temperature

• Wash and add biotin-conjugated WDR35 antibody (1:1000 dilution), incubate for 1 hour

• Add streptavidin-HRP conjugate (1:5000), incubate for 30 minutes

• Develop with TMB substrate and measure absorbance at 450nm

This protocol consistently achieves detection sensitivity of 0.062 ng/ml with minimal interference from other biological factors . The assay range of 0.156-10 ng/ml is suitable for most biological samples, including serum, plasma, and cell culture supernatants .

How can researchers optimize immunofluorescence protocols for visualizing ciliary WDR35 using biotin-conjugated antibodies?

WDR35 localization to cilia presents unique challenges due to its dynamic trafficking and relatively low abundance. For optimal IF visualization:

• Fixation method: 4% PFA for 10 minutes preserves ciliary structures while maintaining epitope accessibility

• Permeabilization: Mild detergents (0.1% Triton X-100) for 5 minutes prevent ciliary structure disruption

• Blocking: 4% goat serum in PBS-T for 60 minutes minimizes background

• Primary incubation: Biotin-conjugated WDR35 antibody at 1:200-1:800 dilution overnight at room temperature

• Detection: Streptavidin-fluorophore conjugate (1:1000) for 1 hour at room temperature

• Co-staining markers: Include acetylated α-tubulin (ciliary axoneme) and γ-tubulin (basal body) for context

This protocol has been successfully employed to visualize WDR35 in multiple cell types, including MDCK cells and hTERT-RPE1 cells , which are widely used ciliary models.

What controls should be included when using biotin-conjugated WDR35 antibodies in experimental designs?

Rigorous experimental design requires appropriate controls to ensure data validity:

• Negative controls:

  • Secondary-only controls (omitting primary antibody) to assess non-specific binding

  • Isotype controls (matched concentration of irrelevant biotin-conjugated IgG)

  • WDR35 knockout or knockdown samples where available

• Positive controls:

  • Cells with known WDR35 expression (MDCK cells, hTERT-RPE1 cells)

  • Recombinant WDR35 protein standards for quantitative assays

  • Rescue experiments using WDR35-EmGFP in WDR35-/- cells

• Technical validation:

  • Peptide competition assays to confirm epitope specificity

  • Correlation with mRNA expression data

  • Cross-validation with alternative detection methods

Inclusion of these controls enables confident interpretation of experimental results and provides critical evidence for reviewers and readers of published work.