DCHS1 Antibody, Biotin conjugated

What is DCHS1 and why is it important in research?

DCHS1 (dachsous cadherin-related 1) is a large calcium-dependent cell-adhesion protein with significant research importance. In humans, the canonical protein has 3298 amino acid residues and a mass of 346.2 kDa, with primary subcellular localization in the cell membrane . DCHS1 functions in multiple developmental processes, including:

• Cell adhesion in fibroblasts (not expressed in melanocytes or keratinocytes)

• Regulation of cell migration involved in valve formation

• Neuroprogenitor cell proliferation and differentiation

• Planar cell polarity (PCP) mechanisms affecting tissue morphogenesis

The protein undergoes post-translational modifications, notably glycosylation . DCHS1 has been implicated in several pathological conditions, particularly mitral valve prolapse (MVP), making it an important research target for cardiovascular studies . Additional associations include Van Maldergem syndrome, bipolar disorder, neuronal heterotopia, and defects in kidney and bone development .

What are the key applications for biotin-conjugated DCHS1 antibodies?

Biotin-conjugated DCHS1 antibodies offer versatility across multiple applications:

• ELISA: The predominant application for biotin-conjugated DCHS1 antibodies, allowing for sensitive protein detection with streptavidin-based reporter systems

• Immunofluorescence (IF): Enables visualization of DCHS1 localization in tissues and cells

• Immunohistochemistry (IHC): Particularly useful for examining DCHS1 expression in paraffin-embedded tissue sections

• Co-immunoprecipitation: Facilitates investigation of protein-protein interactions, especially valuable for studying the DCHS1-LIX1L-SEPT9 complex

• Western blotting: Allows detection of DCHS1 protein (~350 kDa) in tissue and cell lysates

The biotin conjugation enables signal amplification through streptavidin binding, which is particularly advantageous when studying proteins with lower expression levels or in complex tissue environments.

What are the recommended storage and handling conditions for biotin-conjugated DCHS1 antibodies?

Optimal storage and handling conditions for biotin-conjugated DCHS1 antibodies typically include:

• Storage temperature: Store at -20°C for 12 months; some preparations may be stored at -80°C for longer periods

• Storage buffer: Commonly supplied in aqueous buffered solutions containing:

  • 0.01M TBS or PBS (pH 7.4)

  • 50% Glycerol as a cryoprotectant

  • 0.03% Proclin300 as a preservative

  • 1% BSA for stability

• Avoid repeated freeze-thaw cycles: Aliquoting is recommended to maintain antibody integrity

• Working dilutions:

  • ELISA: 1:500-1000

  • IF/IHC: 1:200-400

  • Western blot: 1:500-1000

When preparing working dilutions, use fresh, sterile buffers free of contaminants to prevent non-specific binding or interference with the biotin-streptavidin interaction.

How can biotin-conjugated DCHS1 antibodies be optimized for studying mitral valve prolapse (MVP) mechanisms?

Optimizing biotin-conjugated DCHS1 antibodies for MVP research requires careful experimental design:

Tissue sample preparation considerations:

• For human or mouse mitral valve tissues, optimal fixation is critical—4% paraformaldehyde preserves epitope accessibility better than formalin for these studies

• When examining developmental stages, consider the temporal expression patterns of DCHS1, which shows distinct localization at the AV junction at 54 and 72 hours post-fertilization in zebrafish models

Experimental design recommendations:

• Co-localization studies: Pair biotin-conjugated DCHS1 antibodies with fluorophore-conjugated antibodies against LIX1L and SEPT9 to investigate the complex in valve tissues

• Genetic model integration: When working with DCHS1 mutant mouse models (especially DCHS1+/− and DCHS1−/−), ensure antibody validation against wild-type controls to confirm specificity

• Protein stability assessments: Given that DCHS1 mutations (p.P197L, p.R2513H, p.R2330C) significantly reduce protein half-life (wild-type: 5.8 hours vs. mutant: 1.6 hours), design time-course experiments accounting for this rapid degradation

Technical approach:

• Use streptavidin-coupled reporter systems with signal amplification for detecting low levels of DCHS1 in affected tissues

• For three-dimensional valve structure analysis, combine antibody staining with 3D reconstruction techniques to capture morphological changes in anterior and posterior mitral leaflets

What are the molecular interactions of DCHS1 and how can biotin-conjugated antibodies help elucidate these pathways?

DCHS1 participates in complex molecular interactions that can be effectively studied using biotin-conjugated antibodies:

Key interaction partners:

• LIX1L: Yeast two-hybrid screens identified LIX1L as an abundant binding partner of DCHS1, interacting with amino acids 3130-3191 of the DCHS1 cytoplasmic tail

• SEPT9: Forms part of the DCHS1-LIX1L-SEPT9 (DLS) complex, with SEPT9 expression dependent on the presence of both DCHS1 and LIX1L in developing valves

• FAT4: Functions as a receptor for DCHS1 in heterotypic interactions affecting planar cell polarity pathways

Experimental approaches using biotin-conjugated DCHS1 antibodies:

Research findings to consider:

• Expression of DCHS1 in HEK293T cells fails to result in detectable protein when transfected alone or with SEPT9, but co-expression with LIX1L enables robust expression of both DCHS1 and LIX1L

• The complex shows synergistic genetic interactions, as compound heterozygote mice (DCHS1+/−; LIX1L+/−) develop significantly enlarged valve leaflets compared to single heterozygotes

What controls and validation steps are essential when using biotin-conjugated DCHS1 antibodies?

Rigorous controls and validation are critical for ensuring reliable results with biotin-conjugated DCHS1 antibodies:

Essential controls:

• Specificity controls:

  • Include DCHS1 knockout or knockdown samples to verify antibody specificity

  • Test reactivity against related proteins (e.g., other cadherin family members like CDH19, CDH25)

  • Validate across species if working with animal models, as DCHS1 orthologs exist in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

• Biotin-specific controls:

  • Include biotin blocking steps to prevent endogenous biotin interference

  • Use streptavidin-only controls without primary antibody to assess background binding

  • Consider high-biotin sample interference in experimental design, particularly in tissue samples with naturally high biotin content

• Technical validation:

  • Perform peptide competition assays using synthetic peptides corresponding to the DCHS1 immunogen

  • For antibodies targeting specific DCHS1 domains, verify correct molecular weight detection (~350 kDa)

  • Test antibody performance against both wild-type and mutant DCHS1 proteins, especially when studying MVP-associated mutations

Validation workflow table:

How can researchers optimize biotin-conjugated DCHS1 antibodies for studying the DCHS1-LIX1L-SEPT9 complex?

To effectively study the DCHS1-LIX1L-SEPT9 (DLS) complex using biotin-conjugated DCHS1 antibodies, researchers should implement the following optimization strategies:

Experimental design considerations:

• Developmental timing: The DLS complex shows temporal expression patterns during valve morphogenesis from embryonic day 15.5 (E15.5) to postnatal day zero (P0), with localization changing to valve and myocardial endothelium by four months of age

• Cellular localization: Expression is primarily restricted to endothelial cells and mesenchymal cells within the atrialis and valve tips, regions known to express high levels of proteoglycans

• Protein stability factors: LIX1L is crucial for DCHS1 stability, as DCHS1 expression is undetectable when transfected alone or with SEPT9, but robust when co-expressed with LIX1L

Optimized protocol elements:

• Tissue preparation: Use gentle fixation methods that preserve membrane protein integrity and epitope accessibility

• Antigen retrieval: Optimize for cadherin detection in formalin-fixed tissues using citrate buffer (pH 6.0)

• Signal amplification: Employ streptavidin-HRP or streptavidin-fluorophore systems with tyramide signal amplification if needed

• Background reduction: Include avidin-biotin blocking steps to reduce non-specific binding

Advanced applications:

Research indicates that compound heterozygosity of DCHS1 and LIX1L results in significant leaflet enlargement compared to single heterozygotes or wildtype controls, particularly affecting the anterior leaflet volume and width in the mid and tip regions .

What are the technical considerations for using biotin-conjugated DCHS1 antibodies in multi-color immunofluorescence studies?

Successful multi-color immunofluorescence studies using biotin-conjugated DCHS1 antibodies require careful technical planning:

Protocol optimization:

• Antibody order and detection system:

  • Apply biotin-conjugated DCHS1 antibody first or last in the staining sequence to minimize cross-reactivity

  • If using multiple biotin-conjugated antibodies, consider sequential detection with intervening blocking steps

  • Select appropriate streptavidin conjugates that have minimal spectral overlap with other fluorophores used

• Signal strength and specificity considerations:

  • Implement tiered signal amplification using streptavidin-HRP and tyramide signal amplification for weaker signals

  • For multi-channel imaging, balance signal strengths across channels to prevent bleed-through

  • Test for and eliminate potential cross-reactivity between detection systems

• Tissue and fixation considerations:

  • When examining cardiac tissues for DCHS1 localization, consider that paraformaldehyde fixation preserves membrane proteins better than formalin

  • For DCHS1 detection in valve tissues, optimize antigen retrieval methods to expose epitopes while maintaining tissue morphology

Common technical challenges and solutions:

Recommended fluorophore combinations for multi-color imaging with biotin-DCHS1:

• Streptavidin-Cy5 (for DCHS1) + Alexa Fluor 488 (for LIX1L) + Alexa Fluor 555 (for SEPT9)

• Consider spectral imaging and unmixing for closely overlapping fluorophores

How do different biotinylation methods affect DCHS1 antibody performance in experimental applications?

The choice of biotinylation method can significantly impact the performance of DCHS1 antibodies:

Comparison of biotinylation approaches:

Performance considerations for DCHS1 antibodies:

• Epitope accessibility: Since DCHS1 is a large membrane protein (346.2 kDa) with multiple functional domains, biotinylation methods should be selected to preserve the specific epitope recognition

• Degree of labeling optimization:

  • Excessive biotinylation can reduce antibody activity and increase non-specific binding

  • For DCHS1 detection in complex tissues like heart valves, a biotin:antibody ratio of 4-8:1 typically provides optimal signal-to-noise ratio

• Application-specific recommendations:

  • For ELISA: NHS-biotin conjugation is generally sufficient

  • For immunohistochemistry of valve tissues: Carefully controlled NHS-biotinylation or site-directed methods to preserve epitope recognition

  • For co-IP studies of DCHS1-LIX1L-SEPT9 complex: Mild biotinylation conditions to prevent disruption of antibody-antigen interactions

• Validation metrics: When comparing different biotinylation methods, evaluate:

  • Signal-to-noise ratio in target tissues

  • Specificity (binding to DCHS1 vs. related cadherins)

  • Sensitivity (detection limit for DCHS1)

  • Reproducibility across multiple experimental conditions

Technical note: For studies of DCHS1 mutations associated with mitral valve prolapse, consider that protein stability is significantly reduced in mutant versions (p.P197L, p.R2513H, p.R2330C), which may necessitate more sensitive detection methods regardless of biotinylation approach .

What experimental design factors should be considered when studying DCHS1 protein dynamics using biotin-conjugated antibodies?

Designing experiments to study DCHS1 protein dynamics requires careful consideration of several factors:

Critical experimental parameters:

• Protein half-life considerations:

  • Wild-type DCHS1 protein has a half-life of approximately 5.8 hours

  • Disease-associated mutant variants (p.P197L/p.R2513H) have dramatically reduced half-life of 1.6 hours

  • p.R2330C variant shows even faster degradation (t₁/₂ = 0.46 hours vs. 1.73 hours in controls)

  • Time-course experiments should accommodate these different degradation rates

• Protein complex stability factors:

  • DCHS1 expression is undetectable when transfected alone or with SEPT9

  • Co-expression with LIX1L is necessary for robust DCHS1 expression and detection

  • Experimental designs should account for this dependency on LIX1L for stable expression

• Developmental timing considerations:

  • DCHS1 expression shows temporal specificity during valve development

  • In zebrafish models, expression is predominant at the AV junction at 54 and 72 hours post-fertilization

  • In mouse models, expression patterns change from embryonic day 15.5 (E15.5) to postnatal day zero (P0) and further by 4 months of age

Recommended experimental approaches:

Data interpretation guidelines:

• When quantifying DCHS1 expression using biotin-conjugated antibodies, normalize to appropriate housekeeping proteins

• Consider that DCHS1 expression is primarily in fibroblasts but not in melanocytes or keratinocytes

• For mutation studies, remember that p.R2513H markedly reduces protein levels compared to p.P197L

• In genetic interaction studies, observe that compound heterozygotes (DCHS1+/−; LIX1L+/−) show synergistic effects not seen in single heterozygotes