CBY1 Antibody, HRP conjugated
Description
Molecular and Functional Overview
CBY1 is a 14 kDa protein encoded by the CBY1 gene (UniProt: Q9Y3M2) that regulates Wnt/β-catenin signaling by competitively inhibiting β-catenin’s interaction with TCF/LEF transcription factors . It also modulates intracellular trafficking and cellular differentiation processes .
The HRP-conjugated CBY1 antibody targets amino acids 41–126 of the human CBY1 protein. HRP conjugation allows enzymatic signal amplification, where HRP catalyzes chromogenic substrates like TMB or ABTS, producing detectable colorimetric or chemiluminescent outputs .
3.1. Role in Wnt/β-Catenin Pathway Studies
CBY1’s interaction with β-catenin and DZIP1 has been critical in studying cardiac valve development. In murine models, disruption of the DZIP1-CBY1 complex leads to aberrant β-catenin nuclear localization, causing myxomatous valve degeneration . The HRP-conjugated antibody facilitates quantitative assessment of CBY1 expression levels in such studies .
3.2. Diagnostic and Mechanistic Assays
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ELISA: The antibody’s HRP label enables high-throughput quantification of CBY1 in biological samples, with recommended dilutions of 1:1,000–1:5,000 .
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Western Blot: Detects CBY1 at ~30–35 kDa (observed molecular weight) , though its use in WB requires optimization due to potential buffer incompatibilities .
Technical Considerations
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Buffer Compatibility: Avoid amine-containing buffers (e.g., Tris) during conjugation, as they interfere with HRP activity .
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Sodium Azide Warning: The preservative sodium azide (0.02%) is toxic; handle with protective equipment .
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Validation: Cross-reactivity with mouse CBY1 is predicted but requires empirical confirmation .
Comparative Insights
A 2025 study identified a rare DZIP1 mutation disrupting its interaction with CBY1, underscoring the antibody’s utility in characterizing CBY1-protein complexes . Parallel work using unconjugated CBY1 antibodies (e.g., Proteintech 12239-1-AP) highlights the HRP conjugate’s advantage in reducing assay steps .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- FAM92 proteins interact with Cby1 to promote ciliogenesis via regulation of membrane-remodeling processes. PMID: 27528616
- Chronic Cby1 knockdown in colon cancer cells may counteract tumor progression by promoting the mesenchymal-to-epithelial transition process. PMID: 28107095
- Cby's C-terminal domain alone binds to TC-1 with significantly greater affinity compared to full-length Cby, implying that target binding of the coiled-coil domain is affected by the flanking disordered regions. PMID: 27082063
- CBY1 downregulation in CML comes from reduced protein stability when bound to 14-3-3sigma adapter protein. Dissociation raises CBY1 protein levels by enhancing its stability. The ubiquitin proteasome system reduces 14-3-3sigma-bound CBY1 stability by SUMOylation. PMID: 26147002
- A study revealed that expression of Cby protein was strongly downregulated in laryngeal squamous cell carcinoma (LSCC) tumor tissues in comparison to normal laryngeal mucosa samples. PMID: 25175341
- The impact of BCR-ABL1 on Chibby1, a beta catenin antagonist involved in cell differentiation and transformation PMID: 24339928
- Stable expression of CBY1 drives beta catenin cytoplasmatic translocation and impairs beta catenin signaling in BCR-ABL + cells. PMID: 23707389
- Results from the association analysis suggest that common variation in CBY is not a cause for obesity in the Belgian population. PMID: 23645032
- C-terminal polycystin-2 influences the interaction with PIGEA14 PMID: 23838289
- Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx. PMID: 22911743
- The N-terminal portion of the CBY1 is unstructured in solution, but the C-terminal half forms a coiled-coil structure. PMID: 21182262
- Chibby and clusterin were co-immunoprecipitated with NBPF1. PMID: 20096688
- These findings unravel the molecular basis through which a combinatorial action of Cby and 14-3-3 proteins controls the dynamic nuclear-cytoplasmic trafficking of beta-catenin. PMID: 19940019
- Chibby inhibits beta-catenin-mediated transcriptional activation by competing with Lef-1 to bind to beta-catenin PMID: 12712206
- PIGEA-14 plays an important role in regulating the intracellular location of polycystin-2 PMID: 15194699
- As no somatic mutation was detected in C22orf2 in 36 colorectal tumour DNA, our results do not support the implication of Chibby as a tumour suppressor in colorectal carcinogenesis. PMID: 15245581
- Chibby is not likely to promote colorectal carcinoma tumor development or progression. PMID: 16570344
- CBY promotes adipocyte differentiation through inhibition of beta catenin signaling. PMID: 17403895
- Intrinsically disordered TC-1 interacts with Cby via its transient helical structure PMID: 17905836
- 14-3-3epsilon and 14-3-3zeta are identified as Cby-binding partners. PMID: 18573912
- Genes including CHIBBY involved in pediatric ependymomas. PMID: 18663750
- Alanine substitutions of two or more of four critical leucine residues within the C-terminal heptad repeats eliminate the Cby-Cby interaction. PMID: 19435523
Q&A
How do buffer composition and antibody purity influence HRP-antibody conjugation efficiency and specificity?
Buffer components critically affect conjugation outcomes. For optimal results, buffers should avoid nucleophilic additives like Tris (>50 mM), BSA (>0.1%), or sodium azide, which compete with conjugation reactions . Antibody purity (>90%) is essential to minimize non-specific binding during conjugation. Impurities such as glycine or proclin may interfere with crosslinking reagents .
| Parameter | Recommended Level | Impact on Conjugation |
|---|---|---|
| pH | 6.5–8.5 | Maintains antibody/HRP stability |
| Glycerol | <50% | Prevents viscosity interference |
| BSA/Gelatin | <0.1% | Reduces non-specific binding |
Methodological Insight: Use Lightning-Link® kits for rapid conjugation but confirm buffer compatibility. For high-activity HRP, employ SoluLINK® technology with pre-activated 4-formylbenzamide (4FB)-HRP to form stable hydrazone bonds under mild pH conditions .
What are the advantages and limitations of direct vs. indirect detection using HRP-conjugated CBY1 antibodies?
Optimization Strategy:
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Direct: Use LifeXtend™ stabilizer to mitigate HRP activity loss .
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Indirect: Validate secondary antibodies via CRISPR knockout (KO) models to confirm specificity (e.g., MOR or PKCα KO cells) .
How do traditional vs. modern conjugation methods impact HRP activity and antibody affinity?
Traditional methods like reductive amination or SMCC/2-MEA activation reduce HRP activity (30–50%) and antibody avidity due to harsh oxidation or disulfide bond reduction . Modern SoluLINK® bioconjugation preserves HRP activity (>250 U/mg) by employing hydrazine-aldehyde chemistry under gentle pH (6.0–7.4), avoiding reducing agents .
| Method | HRP Activity Retention | Antibody Affinity Impact |
|---|---|---|
| Reductive amination | 50–70% | Moderate reduction |
| SoluLINK® | >90% | Minimal impact |
Application Note: For phosphospecific CBY1 antibodies, prioritize methods that avoid harsh chemicals to retain epitope recognition .
What experimental controls are essential to validate HRP-conjugated CBY1 antibody specificity in western blotting?
Key controls include:
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KO Cell Lines: Use CRISPR-edited cells lacking CBY1 to confirm absence of signal .
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Blocking Peptides: Compete with the antibody’s epitope to verify antigen-specific binding.
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Secondary Antibody Blots: Exclude non-specific HRP reactivity by omitting primary antibody.
Example Workflow:
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Lyse WT and CBY1-KO cells.
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Load lysates on SDS-PAGE.
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Probing with HRP-conjugated CBY1 should show signal only in WT lanes .
How should conjugates be stored to maximize stability and prevent activity loss?
Store HRP-conjugated antibodies at 4°C in PBS with 50% glycerol and 0.1% BSA. Avoid freeze-thaw cycles and exposure to light. For long-term stability, use LifeXtend™ stabilizer to counteract temperature-induced degradation and dilution effects .
| Storage Condition | Stability Impact |
|---|---|
| 4°C, glycerol/BSA | Optimal (months) |
| Room temperature | Activity halves in weeks |
Troubleshooting: If signal diminishes, re-quantify conjugate using TMB/ABTS assays and adjust dilution ratios .
What strategies mitigate background noise in HRP-based assays using CBY1 antibodies?
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Primary Antibody Optimization: Use high-affinity recombinant antibodies (Kd <1 nM) to reduce non-specific binding .
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Blocking Buffers: Employ casein-based blockers instead of BSA to minimize cross-reactivity.
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Conjugate Purity: Purify conjugates via spin columns to remove unreacted HRP (>90% purity) .
Advanced Approach: Perform sequential blocking (e.g., 5% milk → 1% BSA) to saturate non-specific sites in IHC applications .
How does antibody isotype affect HRP conjugation and assay performance?
IgG subclasses differ in hinge-region disulfide bonds and Fc domains, influencing conjugation efficiency. For example, IgG1 and IgG4 are more stable during maleimide-thiol coupling than IgG2 due to fewer cysteines .
Recommendation: Use IgG1 for consistent conjugation ratios (2–4 HRP per antibody). Validate with SEC-HPLC to confirm monomer integrity .
What are the critical parameters to monitor when scaling up HRP-antibody conjugation?
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Molar Ratio: Maintain 1:2–1:4 antibody-to-HRP ratio to avoid polymerization.
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Reaction Time: Incubate 2–4 hours at room temperature for optimal coupling.
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Purification: Use scavenger resins to remove excess HRP, ensuring <10% unreacted enzyme .
Quality Control: Analyze conjugates via SDS-PAGE or SEC-HPLC to confirm molecular weight and purity .
How do you address conflicting data between western blot and immunohistochemistry using HRP-conjugated CBY1 antibodies?
Discrepancies often arise from epitope accessibility or sample fixation. For WB, denaturing conditions may expose epitopes masked in IHC. Validate using orthogonal methods:
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MS Analysis: Confirm CBY1 presence in lysates.
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Epitope Mapping: Use peptide arrays to identify conformational vs. linear epitopes.
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Antibody Competition: Block with recombinant CBY1 to confirm specificity .
What advanced techniques improve temporal resolution in HRP-based signaling studies?
For dynamic processes (e.g., phosphorylation cascades), combine HRP-conjugated CBY1 with:
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Live-Cell Imaging: Use HRP-TMB substrate for real-time monitoring.
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Single-Cell Analysis: Pair with microfluidic devices to track signaling kinetics.
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Multiplexing: Use HRP variants with distinct substrates (e.g., TMB vs. ABTS) for simultaneous detection .
Example: Track CBY1 activation kinetics post-stimulus using HRP-conjugated antibodies in a time-course ELISA .
