ZBED3 Antibody, HRP conjugated

What is ZBED3 and what are its primary biological functions?

ZBED3 (Zinc-finger BED domain-containing 3) is a cytoplasmic protein that functions as a positive regulator in the canonical Wnt/β-catenin signaling pathway by stabilizing cytoplasmic β-catenin . It plays several important roles including:

• Involvement in transcription activation of Wnt target gene expression

• Regulation of symmetric division of blastomeres during early embryogenesis via mitotic spindle positioning and F-actin filament network organization

• Modulation of cytoskeletal dynamics and cytoplasmic lattice formation to regulate cellular organelle distribution

• Association with insulin resistance in humans, functioning as a potential cytokine

Recent research has identified ZBED3 as a novel secreted protein, suggesting its potential role as a signaling molecule beyond intracellular functions .

How does ZBED3 interact with the Axin protein?

ZBED3 interacts directly with Axin, a scaffold protein involved in β-catenin phosphorylation and degradation. This interaction:

• Was confirmed through yeast two-hybrid screening, co-immunoprecipitation experiments in mammalian cells, and in vitro pulldown assays

• Involves a PPPPSPT motif in ZBED3 that is crucial for binding to Axin

• Requires phosphorylation of serine and threonine residues within this motif for optimal binding

• Primarily occurs in the cytoplasm, where both endogenous ZBED3 and Axin co-localize

When ZBED3 binds to Axin, it appears to inhibit Axin's ability to promote β-catenin degradation, thereby activating Wnt signaling pathways and leading to cytosolic β-catenin accumulation .

What are the primary applications for ZBED3 antibodies in research?

ZBED3 antibodies have several key applications in research settings:

• Western blotting for detection and quantification of ZBED3 protein expression in cell or tissue lysates

• Immunoprecipitation for studying protein-protein interactions involving ZBED3

• ELISA assays for quantitative measurement of ZBED3 in biological samples

• Immunofluorescence for visualizing subcellular localization of ZBED3

These applications allow researchers to investigate ZBED3's role in Wnt signaling, insulin resistance, and embryonic development. ZBED3 antibodies are particularly valuable for studying metabolic disorders, as ZBED3 levels have been found to correlate with insulin resistance markers .

What is the significance of HRP conjugation in ZBED3 antibodies?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages when working with ZBED3 antibodies:

• Enables sensitive colorimetric detection in ELISA assays through enzyme-substrate reactions resulting in measurable color changes

• Allows for quantitative measurement of ZBED3 concentrations when used in sandwich ELISA formats

• Provides signal amplification due to the catalytic nature of the enzyme, enhancing detection sensitivity

• Facilitates detection in Western blotting protocols without requiring secondary antibody incubation steps

In ELISA protocols, HRP-conjugated antibodies specific to ZBED3 are typically used in conjunction with avidin-biotin systems for maximum sensitivity, allowing detection ranges of 0.312-20ng/mL for human ZBED3 .

How should I design experiments to study ZBED3's role in insulin resistance?

Based on current methodologies, a comprehensive approach to studying ZBED3's role in insulin resistance should include:

• Quantification of circulating ZBED3 levels:

  • Use ELISA to measure plasma ZBED3 concentrations in subjects with varying glucose tolerance (normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes)

  • Correlate ZBED3 levels with established insulin resistance markers (BMI, WHR, FAT%, blood pressure, FBG, TG, HbA1c, FIns, HOMA-IR)

• Tissue expression analysis:

  • Compare ZBED3 mRNA and protein expression in muscle and adipose tissue between control subjects and those with T2DM

  • Use real-time PCR for mRNA expression and Western blotting for protein expression

• In vitro regulation studies:

  • Investigate how glucose and insulin concentrations affect ZBED3 secretion

  • Examine ZBED3's effects on insulin signaling by measuring insulin-induced IR and Akt phosphorylation

Cross-sectional and interventional study designs should be employed to establish causative relationships between ZBED3 expression and insulin resistance parameters .

What are optimal conditions for immunoprecipitation of ZBED3 complexes?

For successful immunoprecipitation of ZBED3 and its interacting partners (such as Axin), the following methodology has been validated:

• Cell preparation:

  • Harvest approximately 120 million cells (e.g., NIH3T3) by scraping from culture dishes

  • Lyse cells in buffer containing 0.5% Nonidet P-40, 10% glycerol, 138 mM NaCl, 20 mM Tris-HCl, pH 8.0

  • Include phosphatase inhibitors (10 mM NaF, 2 mM NaVO₄, 1 mM pyrophosphoric acid) and protease inhibitors

• Immunoprecipitation procedure:

  • Clear lysates by centrifugation at 14,000 rpm for 15 minutes

  • Divide supernatant into aliquots and add appropriate antibodies (anti-ZBED3 or anti-Axin)

  • Add Protein A/G plus-agarose and incubate for 3 hours at 4°C

  • Wash immunoprecipitates three times with lysis buffer

  • Denature in SDS sample buffer at 95°C for 10 minutes before SDS-PAGE analysis

This protocol allows effective isolation of ZBED3-containing protein complexes while preserving critical protein-protein interactions for downstream analysis .

How can I quantitatively assess ZBED3 concentrations in clinical samples?

For precise quantification of ZBED3 in clinical samples, a sandwich ELISA approach using HRP-conjugated detection systems offers the best sensitivity and reproducibility:

• ELISA procedure:

  • Use microplates pre-coated with capture antibody specific to ZBED3

  • Add samples or standards to appropriate wells, followed by biotin-conjugated detection antibody

  • Add avidin-HRP conjugate and incubate

  • Add TMB substrate solution and measure color change spectrophotometrically at 450nm ± 10nm

• Data analysis:

  • Construct a standard curve by plotting mean optical density against known ZBED3 concentrations

  • Determine sample concentrations by comparing OD values to the standard curve

  • Account for any dilution factors in the final calculation

The detection range for human ZBED3 using this method is typically 0.312-20ng/mL, with standard curve concentrations of 20, 10, 5, 2.5, 1.25, 0.625, and 0.312 ng/mL .

What mechanisms regulate ZBED3's effects on Wnt/β-catenin signaling?

ZBED3 regulates Wnt/β-catenin signaling through several molecular mechanisms:

• Direct interaction with Axin:

  • ZBED3 binds to Axin through a PPPPSPT motif similar to the PPPP(S/T)PX(T/S) motif in LRP5/6

  • This interaction may sequester Axin away from the β-catenin destruction complex

• Phosphorylation-dependent regulation:

  • Phosphorylation of serine and threonine residues in the PPPPSPT motif of ZBED3 is crucial for Axin binding

  • Mutations of these residues (S to A or T to A) significantly reduce binding affinity

• β-catenin stabilization:

  • Overexpression of ZBED3 leads to accumulation of cytosolic β-catenin, similar to Wnt3a treatment

  • This stabilization activates downstream Wnt target gene expression

• Regulatory feedback:

  • RNAi-mediated knockdown of endogenous ZBED3 decreases Wnt/β-catenin signaling, confirming its physiological role in this pathway

Understanding these mechanisms is essential for developing potential therapeutic approaches targeting the ZBED3-Axin interaction in diseases associated with aberrant Wnt signaling .

How can I optimize Western blotting protocols for ZBED3 detection?

For optimal Western blot detection of ZBED3, consider the following methodological recommendations:

• Sample preparation:

  • For cellular fractionation studies, include separate cytoplasmic, nuclear, and membrane fractions to accurately determine ZBED3 localization

  • Use phosphatase inhibitors (NaF, NaVO₄, pyrophosphoric acid) to preserve phosphorylation states

• Antibody selection and optimization:

  • Use validated antibodies with confirmed specificity for ZBED3

  • For immunodetection, an optimal antibody concentration of 1 μg/mL has been validated for ZBED3 detection

  • Predicted band size for ZBED3 is approximately 25 kDa

• Controls:

  • Include positive controls such as A-20 cell lysate, which has been validated for ZBED3 detection

  • Consider using ZBED3-overexpressing cells as additional positive controls

• Signal enhancement:

  • For HRP-conjugated detection systems, optimize substrate exposure times for maximum sensitivity without background issues

  • Consider using enhanced chemiluminescence systems for low-abundance detection

These optimizations will help ensure specific and sensitive detection of ZBED3 protein in experimental samples.

What approaches can address inconsistent ELISA results when measuring ZBED3?

When troubleshooting ELISA assays for ZBED3 quantification, consider these methodological approaches:

• Standard curve optimization:

  • Plot the standard curve on log-log graph paper with ZBED3 concentration on the y-axis and absorbance on the x-axis

  • Use appropriate curve-fitting software (e.g., curve expert 1.30) for accurate interpolation

  • Ensure standards cover the appropriate concentration range (0.312-20ng/mL for human ZBED3)

• Sample handling:

  • Account for potential matrix effects by appropriate sample dilution

  • Ensure consistent sample processing to minimize variability

  • Use multiple sample dilutions to verify results fall within the linear range of the assay

• Technical considerations:

  • Standardize pipetting technique and washing procedures as these can significantly affect assay performance

  • Control temperature effects during incubation steps

  • Run all samples in duplicate or triplicate to identify outliers

• Data analysis:

  • Calculate the concentration by comparing OD values to the standard curve

  • Remember to multiply by any dilution factors used during sample preparation

  • Consider plotting individual calibration curves for each assay plate

Following these guidelines will improve reproducibility and accuracy when measuring ZBED3 concentrations in research samples.

How does ZBED3 expression correlate with metabolic disease parameters?

Research has established several significant correlations between ZBED3 expression and metabolic disease markers:

• Circulating ZBED3 levels correlate with:

  • Positive correlation with BMI, WHR, FAT%, blood pressure, FBG, TG, HbA1c, FIns, and HOMA-IR

  • Inverse correlation with HDL-C levels

  • Higher levels in individuals with impaired glucose tolerance (IGT) and newly diagnosed type 2 diabetes (nT2DM) compared to those with normal glucose tolerance (NGT)

• Tissue expression patterns:

  • ZBED3 mRNA and protein expression in muscle and adipose tissue are significantly elevated in both db/db mice and T2DM patients

  • These expression levels correlate with insulin resistance parameters

• Regulation by metabolic factors:

  • Glucose exhibits a concentration-dependent effect on ZBED3 release

  • Insulin shows an inhibitory effect on ZBED3 levels

  • ZBED3 suppresses insulin-induced IR and Akt phosphorylation, suggesting a direct role in insulin signaling modulation

These correlations suggest ZBED3 may be a valuable biomarker for metabolic disorders and potentially a therapeutic target for insulin resistance-related conditions .

What role might ZBED3 play in embryonic development research?

ZBED3's functions in embryonic development make it a relevant target for developmental biology research:

• Blastomere division regulation:

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

  • It regulates mitotic spindle central positioning and organization of the F-actin filament network

• Cellular organization control:

  • ZBED3 regulates the distribution of cellular organelles by modulating cytoskeletal dynamics

  • It contributes to cytoplasmic lattice formation, essential for proper embryonic cell architecture

• Wnt signaling in development:

  • As an Axin-interacting protein, ZBED3 modulates Wnt/β-catenin signaling

  • This pathway is crucial for proper embryonic development and tissue patterning

Researchers focusing on embryonic development should consider examining ZBED3 expression patterns during different developmental stages and investigating how disruption of ZBED3 affects embryonic patterning and organogenesis, particularly through its effects on Wnt signaling pathways.