CHRDL1 Monoclonal Antibody

What is CHRDL1 and what are its primary biological functions?

CHRDL1 (also known as Neuralin-1, Neurogenesin-1, or Ventroptin) is a secreted protein that antagonizes BMP4 function by binding to it and preventing its interaction with receptors. Its biological functions include:

• Neural stem cell fate regulation - shifting from gliogenesis to neurogenesis

• Promoting neuronal differentiation by preventing glial fate adoption

• Contributing to dorsoventral axis formation during development

• Involvement in embryonic bone formation

• Modulation of retinal angiogenesis through BMP4 regulation

• Participating in anterior segment eye development

Research demonstrates that CHRDL1 enhances BMP-4-induced phosphorylation of SMAD1/5/9 during osteogenic differentiation, suggesting a complex regulatory mechanism rather than simple antagonism in certain contexts .

How are CHRDL1 monoclonal antibodies validated for research applications?

CHRDL1 monoclonal antibodies undergo extensive validation processes before being recommended for specific applications:

• Target specificity verification: Testing against recombinant human CHRDL1 protein (such as mouse myeloma cell line NS0-derived recombinant human CHRDL1 Glu28-Cys457) using direct ELISAs and Western blots

• Cross-reactivity assessment: Evaluation against similar proteins (e.g., testing against recombinant mouse Chordin-Like 2) to ensure specificity

• Application-specific validation: Testing in various techniques including Western blot, immunohistochemistry, and ELISA

• Species reactivity profiling: Determining which species the antibody effectively recognizes (commonly human and rat for many CHRDL1 antibodies)

Antibodies are typically assigned reactivity profiles indicating whether they have been confirmed to work with specific species and applications, forming the basis of vendor guarantees and reliability assessments .

What are the key considerations when designing experiments using CHRDL1 antibodies?

When designing experiments with CHRDL1 antibodies, researchers should consider:

• Antibody format compatibility: Determine if the antibody is compatible with your application - many CHRDL1 antibodies are validated for Western blot (1 μg/mL) but may require optimization for other applications

• Sample preparation: CHRDL1 has multiple isoforms (4 reported) with an expected mass of 52 kDa - ensure your sample preparation methods preserve the protein structure and epitopes

• Control selection: Include both positive controls (tissues known to express CHRDL1, such as bone formation regions) and negative controls

• Reconstitution protocols: Follow manufacturer recommendations (e.g., reconstituting lyophilized antibodies at 0.5 mg/mL in sterile PBS) to maintain antibody function

• Storage conditions: Adhere to recommended storage protocols (-20 to -70°C for long-term; 2-8°C for reconstituted antibodies for up to 1 month) and avoid repeated freeze-thaw cycles

How does CHRDL1 function in BMP signaling pathways and how can this be experimentally investigated?

CHRDL1's role in BMP signaling demonstrates contextual complexity beyond simple antagonism:

• Dual modulatory effects: While CHRDL1 antagonizes BMP4 by direct binding, research shows that in certain contexts (such as osteogenic differentiation), it can enhance BMP-4-induced SMAD1/5/9 phosphorylation, suggesting context-dependent modulatory functions

• Experimental approaches to investigate these mechanisms include:

  • Phosphorylation assays: Western blots targeting p-Smad1/5/9 levels with and without CHRDL1 overexpression/knockdown

  • Inhibitor studies: Using BMP receptor type I inhibitors (such as LDN-193189) to block CHRDL1-mediated effects

  • Co-immunoprecipitation: To detect direct interactions between CHRDL1 and BMP4

  • Expression analysis: Real-time quantitative PCR measurements of downstream genes (e.g., ALP, COL1A1, OCN) following CHRDL1 manipulation

Research has shown that BMP-4 induces CHRDL1 expression in a time- and dose-dependent manner, creating a feedback loop. This induction can be blocked by BMP inhibitors like LDN-193189, providing a mechanism to experimentally manipulate this pathway .

What methodological approaches are most effective for studying CHRDL1's role in osteogenic differentiation?

To investigate CHRDL1's role in osteogenic differentiation, researchers have successfully employed these methodological approaches:

• Gene modification techniques:

  • Overexpression using vectors (e.g., pLVX-CHRDL1)

  • Knockdown using siRNAs (multiple siRNAs targeting different regions for validation)

  • CRISPR-Cas9 for stable genetic manipulation

• In vitro osteogenic differentiation assays:

  • ALP staining and quantitative analysis

  • Alizarin Red staining for matrix mineralization

  • Gene expression analysis of osteogenic markers (OCN, COL1A1, OPN, ALP, OSX, RUNX2)

• In vivo bone formation models:

  • Transplantation of CHRDL1-modified human BMSCs into nude mice femur defect models

  • Quantification of new bone formation rates

  • Immunohistochemical detection of CHRDL1 expression in new bone formation regions

These approaches have revealed that CHRDL1 overexpression significantly promotes osteogenic differentiation both in vitro and in vivo, while knockdown suppresses this process, suggesting therapeutic potential for bone regeneration applications .

How can CHRDL1 expression be accurately quantified in research samples?

For precise quantification of CHRDL1 expression in research samples, multiple complementary techniques should be employed:

• Protein-level quantification:

  • Western blot analysis using validated monoclonal antibodies (e.g., R&D Systems antibodies at 1 μg/mL concentration)

  • Sandwich ELISA with capture antibody at 2 μg/mL and detector antibody at 0.5 μg/mL

  • Immunohistochemistry for tissue localization and semi-quantitative analysis

• mRNA-level quantification:

  • Real-time quantitative PCR with validated primer pairs

  • RNA-seq for transcriptome-wide expression profiling

  • In situ hybridization for spatial localization in tissues

• Normalization strategies:

  • For Western blots: Densitometric analysis with normalization to housekeeping proteins (GAPDH)

  • For qPCR: Normalization to stable reference genes appropriate for the tissue/cell type under study

  • For immunohistochemistry: Digital image analysis with standardized protocols

When evaluating CHRDL1 expression changes, consider time-course experiments, as studies have shown dynamic expression patterns following stimulation (e.g., BMP-4 induces CHRDL1 expression with peaks at specific timepoints) .

How should experiments be designed to study CHRDL1's role in cancer pathogenesis?

Recent research has identified CHRDL1 as a potential prognostic biomarker in lung adenocarcinoma (LUAD). When designing experiments to study CHRDL1's role in cancer:

• Clinical sample analysis:

  • Compare CHRDL1 expression between tumor and adjacent normal tissues using immunohistochemistry

  • Correlate expression levels with clinicopathologic features (T stage, N stage, treatment response, tumor status)

  • Perform survival analysis stratified by CHRDL1 expression levels

• Mechanistic studies:

  • Investigate potential relationships between CHRDL1 and known cancer pathways through:

    • Gene Set Enrichment Analysis (GSEA) to identify enriched pathways

    • Cell cycle analysis in CHRDL1-manipulated cell lines

    • Immune infiltration analysis to correlate CHRDL1 with immune cell types

• Validation approaches:

  • Use multiple antibody clones to confirm expression patterns

  • Include positive controls (tissues with known high CHRDL1 expression)

  • Employ both protein and mRNA detection methods

  • Validate findings across independent patient cohorts

What controls and validation steps are necessary when using CHRDL1 antibodies in functional studies?

When conducting functional studies with CHRDL1 antibodies, implement these essential controls and validation steps:

• Antibody specificity controls:

  • Use multiple antibody clones targeting different epitopes

  • Include recombinant CHRDL1 protein as a positive control

  • Test against similar family proteins (e.g., Chordin-Like 2) to confirm specificity

  • Include isotype controls to rule out non-specific binding

• Genetic validation approaches:

  • Compare antibody staining in CHRDL1 knockdown/knockout cells

  • Use overexpression systems to confirm detection of increased protein levels

  • Verify antibody detects known isoforms (CHRDL1 has 4 reported isoforms)

• Functional validation strategies:

  • Pair antibody-based detection with functional readouts (e.g., SMAD phosphorylation)

  • Confirm antibody-detected expression changes correlate with functional outcomes

  • Use pathway inhibitors (e.g., LDN-193189) to block CHRDL1-mediated effects

• Technical controls:

  • Titrate antibody concentrations to determine optimal working dilutions

  • Include secondary antibody-only controls to assess background

  • Perform cross-adsorption studies if non-specific binding is suspected

How can researchers effectively manipulate CHRDL1 levels in experimental models?

For effective manipulation of CHRDL1 levels in experimental systems, researchers can employ these strategic approaches:

• Genetic manipulation techniques:

  • Overexpression: Transfection with CHRDL1 expression vectors (e.g., pLVX-CHRDL1) results in significant upregulation at both mRNA and protein levels

  • Knockdown: Multiple siRNAs targeting different regions of CHRDL1 (evaluated studies used three different siRNAs with siRNA3 showing ~70% knockdown efficiency)

  • Knockout: CRISPR-Cas9 for complete elimination of CHRDL1 expression

• Pharmacological modulation:

  • Indirect upregulation: BMP-4 treatment induces CHRDL1 expression in a time- and dose-dependent manner

  • Indirect inhibition: BMP receptor type I inhibitor LDN-193189 (100 nM) significantly decreases CHRDL1 expression

• Recombinant protein approaches:

  • Application of purified recombinant CHRDL1 protein

  • Use of neutralizing antibodies to block endogenous CHRDL1 function

• Verification methods:

  • Confirm expression changes at both mRNA (qPCR) and protein (Western blot) levels

  • Assess timeframe of manipulation effects (studies show siRNA effects lasting up to 10 days)

  • Monitor effects on cell viability (CCK8 assay) to ensure observed effects aren't due to cytotoxicity

How can contradictory findings regarding CHRDL1 function be reconciled in different experimental contexts?

CHRDL1 has shown seemingly contradictory functions in different experimental systems, particularly regarding its relationship with BMP signaling:

• Contextual interpretation framework:

  • Cell/tissue type specificity: CHRDL1 may have different effects in neural tissues versus bone tissues

  • Developmental stage dependency: Functions may differ during embryonic development versus adult tissues

  • Concentration-dependent effects: CHRDL1 may have biphasic effects depending on concentration

  • BMP ligand specificity: While primarily studied as a BMP4 antagonist, CHRDL1 may interact differently with other BMP family members

• Methodological reconciliation:

  • Evaluate differences in experimental models (in vitro cell lines vs. in vivo models)

  • Compare protein detection methods and antibody clones used

  • Assess expression levels achieved in overexpression/knockdown studies

  • Consider temporal dynamics of signaling responses

• Resolution strategies:

  • Perform parallel experiments with standardized protocols across different cell types

  • Use domain-specific mutants to isolate functional regions of CHRDL1

  • Employ pathway-focused approaches with phosphorylation status measurements

Research has demonstrated that while CHRDL1 antagonizes BMP4 in many contexts, it can enhance BMP-4-induced SMAD1/5/9 phosphorylation during osteogenic differentiation, suggesting context-specific modulatory roles .

What are the common technical challenges when using CHRDL1 antibodies and how can they be addressed?

Researchers often encounter these technical challenges when working with CHRDL1 antibodies:

• Detection sensitivity issues:

  • Challenge: Low endogenous expression levels in some tissues

  • Solution: Use signal amplification methods (e.g., tyramide signal amplification), increase antibody concentration, extend incubation times, or use more sensitive detection systems

• Isoform-specific detection:

  • Challenge: CHRDL1 has 4 reported isoforms

  • Solution: Select antibodies that recognize conserved regions across isoforms or use multiple antibodies targeting different epitopes; verify the specific isoform(s) expressed in your experimental system

• Non-specific binding:

  • Challenge: Background signal in Western blots or immunostaining

  • Solution: Optimize blocking conditions (try different blockers like BSA, milk, or commercial blockers), increase washing steps, or use monoclonal antibodies with higher specificity

• Sample preparation considerations:

  • Challenge: Loss of epitope accessibility during fixation

  • Solution: Compare different fixation methods (formalin, methanol, acetone) and optimize antigen retrieval procedures (heat-induced epitope retrieval with citrate or EDTA buffers)

• Storage and handling:

  • Challenge: Antibody degradation affecting performance

  • Solution: Store according to manufacturer recommendations (typically -20 to -70°C), avoid repeated freeze-thaw cycles, prepare single-use aliquots, and use carrier proteins for dilute solutions

How should researchers interpret changes in CHRDL1 expression in relation to BMP signaling activity?

Interpreting the relationship between CHRDL1 expression and BMP signaling requires nuanced analysis:

• Bidirectional relationship assessment:

  • Measure both CHRDL1 levels and downstream BMP signaling markers (p-SMAD1/5/9, BMPR expression)

  • Recognize that BMP-4 induces CHRDL1 expression in a time- and dose-dependent manner, creating a feedback loop

  • Consider the temporal dynamics - immediate versus long-term effects

• Integrated pathway analysis:

  • Use SMAD phosphorylation status as a direct readout of BMP pathway activation

  • Examine expression of BMP target genes (e.g., ID1, ID2) alongside CHRDL1 expression

  • Consider interactions with other BMP regulators (noggin, chordin, follistatin)

• Context-specific interpretation:

  • In osteogenic differentiation: CHRDL1 overexpression enhances BMP-4-induced SMAD1/5/9 phosphorylation and improves bone formation

  • In neural contexts: CHRDL1 may function as a classical BMP antagonist

  • In cancer contexts: Low CHRDL1 correlates with poor prognosis in some cancers

• Experimental verification:

  • Use BMP receptor inhibitors (LDN-193189) to validate CHRDL1-dependent effects

  • Perform rescue experiments to confirm specificity of observed effects

  • Compare effects of direct BMP4 addition versus CHRDL1 manipulation

What are the best practices for using CHRDL1 antibodies in immunohistochemistry of tissue samples?

For optimal results when using CHRDL1 antibodies in immunohistochemistry:

• Tissue preparation optimization:

  • Test multiple fixation protocols (10% neutral buffered formalin is commonly used)

  • Optimize antigen retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Consider tissue-specific modifications based on protein abundance

• Antibody selection and validation:

  • Choose antibodies specifically validated for IHC applications

  • Validate antibody performance using positive control tissues (bone formation regions, lung adenocarcinoma samples)

  • Include negative controls (isotype controls and tissues with low/no CHRDL1 expression)

• Signal detection optimization:

  • Titrate primary antibody concentration to find optimal signal-to-noise ratio

  • Select appropriate detection systems (HRP-DAB, fluorescence) based on expression level

  • Consider signal amplification methods for low-abundance expression

• Quantification strategies:

  • Use digital image analysis software for objective quantification

  • Develop consistent scoring systems (e.g., H-score, proportion scoring)

  • Apply automated algorithms for cellular/subcellular localization analysis

Studies have successfully used IHC to detect CHRDL1 in new bone formation regions of defective femur models and to evaluate its expression in lung adenocarcinoma tissues .

How can CHRDL1 antibodies be effectively utilized in studying its role in immune regulation?

Recent research has identified connections between CHRDL1 and immune regulation, particularly in cancer contexts. To study these relationships:

• Immune infiltration analysis approaches:

  • Use multiplex immunofluorescence to co-localize CHRDL1 with immune cell markers

  • Perform correlation analysis between CHRDL1 expression and immune cell populations

  • Apply computational deconvolution methods to estimate immune cell abundance from expression data

• Functional immune assays:

  • Evaluate T cell activation and proliferation in the presence of CHRDL1

  • Assess cytokine production profiles with and without CHRDL1 manipulation

  • Examine immune checkpoint molecule expression in relation to CHRDL1 levels

• Methodological considerations:

  • Include antibodies against established immune cell markers for co-localization studies

  • Use flow cytometry to quantify immune cell populations in CHRDL1-manipulated systems

  • Apply cytokine arrays or multiplex assays to evaluate secreted factors

• Interpretation framework:

  • Consider tumor microenvironment context when interpreting results

  • Recognize that CHRDL1 may have indirect effects on immune cells through BMP pathway modulation

  • Account for cell-type specific responses when analyzing heterogeneous samples

Research in lung adenocarcinoma has shown that CHRDL1 expression is significantly correlated with 7 kinds of immune cells and may be negatively correlated with Th2 cells, suggesting potential roles in immunotherapy response .