ZBED3 Antibody, Biotin conjugated

What is ZBED3 and why is it significant in cellular biology?

ZBED3 (zinc finger BED-type containing 3) is a 234 amino acid protein with a molecular weight of approximately 25.1 kDa that functions primarily in the cytoplasm before being secreted. Its biological significance stems from its role as a positive regulator in the canonical Wnt/β-catenin signaling pathway, where it stabilizes cytoplasmic β-catenin . ZBED3 is widely expressed across multiple tissue types, with notable expression in bronchus and fallopian tube tissues . The protein plays crucial roles in symmetric division of blastomeres during early embryogenesis through regulation of mitotic spindle positioning and F-actin filament network organization . Additionally, ZBED3 influences cellular organelle distribution by modulating cytoskeletal dynamics and cytoplasmic lattice formation .

What applications are ZBED3 antibodies typically used for?

ZBED3 antibodies, including biotin-conjugated variants, are employed in several immunodetection techniques:

ELISA is particularly valuable for quantitative detection of ZBED3 in human tissue homogenates, cell lysates, and other biological fluids . In standard ELISA protocols, samples are added to microtiter plate wells alongside a biotin-conjugated antibody specific to ZBED3, followed by addition of Avidin conjugated to Horseradish Peroxidase (HRP) . This creates a sandwich complex that enables sensitive detection following substrate addition and spectrophotometric measurement .

How does the biotin conjugation impact ZBED3 antibody functionality?

Biotin conjugation provides significant advantages for ZBED3 detection through enhanced sensitivity and versatility. The biotin-avidin/streptavidin system represents one of the strongest non-covalent biological interactions known, allowing for amplified signal detection when using biotin-conjugated ZBED3 antibodies . The biotin modification facilitates a multi-layered detection approach, where primary binding occurs between the antibody and ZBED3, followed by secondary detection via biotin-avidin binding and enzymatic signal amplification . This system works particularly well in sandwich ELISA formats where the biotin-conjugated antibody serves as the detection antibody while maintaining specific recognition of ZBED3 epitopes. The methodology employs Avidin conjugated to HRP, which binds with high affinity to the biotin moiety, creating a detection complex capable of producing strong colorimetric signals when TMB substrate is added .

What are the optimal storage and handling conditions for biotin-conjugated ZBED3 antibodies?

Biotin-conjugated ZBED3 antibodies require careful handling to maintain their functionality. Standard storage recommendations include:

Long-term storage should follow manufacturer recommendations, with most suppliers suggesting storage at -20°C for unopened/unused antibody . Once reconstituted or diluted, aliquoting is strongly recommended to avoid repeated freeze-thaw cycles that can compromise both the antibody binding capacity and the biotin conjugation. When planning experiments, researchers should prepare working dilutions fresh or store them at 4°C for short durations only.

How should researchers optimize protocols for ELISA using biotin-conjugated ZBED3 antibodies?

Optimizing ELISA protocols with biotin-conjugated ZBED3 antibodies requires methodical adjustment of several parameters:

• Antibody titration: Determine optimal concentrations through serial dilution tests (typically starting at 1:100-1:1000 dilutions)

• Blocking optimization: Test different blocking agents (BSA, milk proteins) to minimize background while maintaining specific signal

• Incubation parameters: Evaluate both time (1-2 hours typical) and temperature (room temperature vs. 37°C) effects on signal-to-noise ratio

• Standard curve: Generate using recombinant ZBED3 protein at concentrations spanning the expected detection range

• Signal development: Optimize substrate (TMB) incubation time to achieve suitable color development without saturation

Most commercial ELISA kits using biotin-conjugated antibodies for ZBED3 detection employ a general workflow where samples are added to microplate wells coated with capture antibody, followed by biotin-conjugated detection antibody and HRP-conjugated avidin . After washing steps, TMB substrate solution is added, and the reaction is terminated with acidic stop solution before measuring absorbance at 450nm ± 10nm .

What cross-reactivity concerns should researchers address when using ZBED3 antibodies?

When selecting ZBED3 antibodies, researchers should carefully evaluate potential cross-reactivity with related proteins:

Some commercially available antibodies are specifically designed to have no cross-reactivity with related proteins such as ZBED1 or ZBED2 . When conducting multi-species studies, researchers should verify the conservation of the target epitope, as ZBED3 orthologs have been reported in various species including mouse, rat, bovine, and chimpanzee . For definitive validation, knockout or knockdown systems provide the most reliable negative controls to confirm antibody specificity.

How can ZBED3 antibodies be used to investigate Wnt/β-catenin signaling pathway dynamics?

ZBED3 antibodies offer powerful tools for dissecting Wnt/β-catenin pathway regulation:

• Co-immunoprecipitation studies: Biotin-conjugated ZBED3 antibodies can pull down protein complexes to analyze ZBED3's interaction with key pathway components like Axin and β-catenin

• Sequential binding assays: Use antibodies to track ZBED3's temporal interaction with pathway proteins during Wnt signaling activation

• Subcellular fractionation: Combine with organelle isolation to examine compartment-specific localization changes upon pathway stimulation

• Phosphorylation state analysis: Couple with phospho-specific antibodies to monitor how ZBED3 influences β-catenin phosphorylation status

Research has established that ZBED3 acts as an Axin-interacting protein, modulating the canonical Wnt/β-catenin signaling pathway . Experimental evidence from RNA interference studies showed that ZBED3 knockdown attenuated Wnt-induced β-catenin accumulation, lymphoid enhancer binding factor-1-dependent luciferase reporter activity, and Wnt gene expression . Western blot analysis revealed significant upregulation of AXIN1, GSK3β, and β-catenin expression following ZBED3 overexpression in 786-O cells, while WNT5α and phosphorylated β-catenin showed reduced expression .

What role does ZBED3 play in cancer biology, and how can antibodies help elucidate these mechanisms?

Recent studies have uncovered ZBED3's complex involvement in cancer biology, particularly in kidney renal clear cell carcinoma (KIRC):

Researchers can use biotin-conjugated ZBED3 antibodies for tissue microarray analysis to correlate expression patterns with clinical outcomes across patient cohorts. Additionally, these antibodies enable mechanistic studies through chromatin immunoprecipitation to identify genomic targets of ZBED3-associated transcriptional complexes in cancer cells.

How can researchers integrate ZBED3 antibody data with functional genomics approaches?

Modern research strategies can combine antibody-based detection with genomic technologies:

• ChIP-seq: Use biotin-conjugated ZBED3 antibodies to identify genome-wide binding sites and correlate with transcriptomic changes

• CRISPR screening: Validate phenotypic outcomes of genetic manipulation using quantitative ZBED3 protein detection

• Proteogenomic integration: Correlate protein expression data from antibody-based assays with RNA-seq and genomic datasets

• Single-cell analysis: Combine with single-cell RNA-seq to correlate protein levels with transcriptional heterogeneity

Studies have identified correlations between ZBED3 expression and specific gene sets in KIRC. For example, research demonstrated median positive correlation with Tcm immune cells and median negative correlation with NK CD56bright cells and cytotoxic cells . Gene Set Enrichment Analysis (GSEA) revealed that ZBED3 has negative regulatory effects on pathways including DNA methylation and telomere stress-induced senescence . Researchers identified top correlated genes with ZBED3 expression (RTL1, SFTPB, KLK1, FOXI1, RPL13AP17, PAEP, SSX1, SERPINB4, KRTAP5-8, HMGA2) that could serve as functional partners in determining cell fate commitment, keratinization, and cytokine activity .

What are common technical challenges when working with biotin-conjugated ZBED3 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with biotin-conjugated antibodies:

When troubleshooting ELISA assays specifically, researchers should verify all reagents are at the correct temperature before use, ensure thorough washing between steps, and confirm that the TMB substrate has not deteriorated . For Western blot applications, despite ZBED3's predicted molecular weight of 25 kDa, the protein often migrates at a higher position in SDS-PAGE , so researchers should be prepared for this potential discrepancy when interpreting results.

How should researchers interpret apparent discrepancies between ZBED3 antibody results and transcriptomic data?

Discrepancies between protein and transcript levels are common in biological systems and require careful interpretation:

• Post-transcriptional regulation: Evaluate potential microRNA or RNA-binding protein involvement in translation regulation of ZBED3

• Protein stability differences: Assess half-life variations under different experimental conditions

• Detection sensitivity thresholds: Consider technical limitations of antibody-based versus sequencing-based detection methods

• Compartmentalization effects: Analyze subcellular fractionation data to account for protein localization changes

• Temporal dynamics: Design time-course experiments to capture lag between transcription and protein expression

For ZBED3 specifically, researchers should consider that it functions both in the cytoplasm and as a secreted protein , which can complicate interpretation of cellular expression data. The biotin-conjugated antibody might detect total cellular ZBED3 in fixed cells but miss secreted fractions in live cell studies. Additionally, ZBED3's involvement in multiple cellular processes including Wnt signaling, cell cycle regulation, and cytoskeletal organization suggests that its expression and post-translational modifications may vary considerably depending on cellular context.

What are the most appropriate statistical approaches for analyzing quantitative ZBED3 protein expression data?

When analyzing quantitative data from biotin-conjugated ZBED3 antibody experiments:

For ELISA-based quantification, researchers should construct standard curves using appropriate regression models (linear, 4-parameter logistic) to accurately calculate ZBED3 concentrations . When interpreting Western blot densitometry data, normalization to appropriate housekeeping proteins is essential. For clinical correlations, such as observed in KIRC studies, researchers should stratify patients based on ZBED3 expression levels (e.g., high vs. low based on median values) when conducting survival analyses .

How can biotin-conjugated ZBED3 antibodies contribute to understanding immune infiltration in cancer?

Recent research has revealed intriguing connections between ZBED3 expression and immune cell infiltration patterns:

• Correlation analysis: ZBED3 expression has demonstrated median positive correlation with central memory T cells (Tcm) in KIRC

• Negative associations: ZBED3 levels show median negative correlation with NK CD56bright cells and cytotoxic cells among 24 distinct immune subtypes in KIRC

• Immune microenvironment mapping: Multispectral imaging with biotin-conjugated ZBED3 antibodies can map spatial relationships between ZBED3-expressing cells and immune populations

• Response prediction: ZBED3 expression patterns might serve as biomarkers for immunotherapy response prediction

Researchers can design multiplex immunofluorescence studies using biotin-conjugated ZBED3 antibodies alongside immune cell markers to investigate spatial relationships within the tumor microenvironment. The differential correlation with specific immune cell types suggests ZBED3 may influence immune recruitment or exclusion mechanisms that could have therapeutic implications .

What is the potential for using ZBED3 as a therapeutic target, and how can antibodies facilitate this research?

While primarily a research tool, ZBED3 antibodies can accelerate therapeutic development:

• Target validation: Confirm ZBED3's causal role in disease processes through antibody-mediated neutralization studies

• Epitope mapping: Identify functional domains critical for ZBED3's interaction with Axin and other Wnt pathway components

• Internalization studies: Assess whether anti-ZBED3 antibodies can be internalized for potential antibody-drug conjugate development

• Mechanistic insights: Elucidate ZBED3's precise role in cell proliferation regulation for identification of druggable nodes

Research demonstrates that ZBED3 overexpression significantly inhibits cell migration in wound healing and transwell experiments . Moreover, CCK-8 assay results show that upregulation of ZBED3 results in decreased cell proliferation . This tumor-suppressive role in KIRC suggests that therapeutic strategies aimed at enhancing ZBED3 activity might have anti-cancer effects. Antibody studies have helped reveal that ZBED3 overexpression leads to substantial upregulation of AXIN1, GSK3β, and β-catenin while reducing WNT5α and phosphorylated β-catenin levels , providing potential mechanistic targets for intervention.

How might ZBED3 research inform our understanding of developmental biology and embryogenesis?

ZBED3's roles extend beyond cancer into fundamental developmental processes:

• Symmetric cell division: ZBED3 plays a role in symmetric division of blastomeres during early embryogenesis

• Cytoskeletal regulation: It influences mitotic spindle positioning and F-actin filament network organization

• Organelle distribution: ZBED3 regulates cellular organelle distribution through cytoskeletal dynamics

• Wnt signaling modulation: Its effects on this pathway have significant implications for development and tissue patterning

Biotin-conjugated ZBED3 antibodies can enable developmental studies through immunofluorescence imaging in embryonic tissues to track expression patterns during critical developmental windows. Time-course studies during embryogenesis could reveal stage-specific functions, while co-localization with cytoskeletal markers might elucidate its role in organizing subcellular architecture essential for proper development. The established role of ZBED3 in regulating Wnt/β-catenin signaling further connects it to key developmental pathways involved in axis formation, tissue specification, and organogenesis.