DCBLD2 Antibody

What is DCBLD2 and why is it significant in research?

DCBLD2 (Discoidin, CUB and LCCL Domain Containing 2), also known as ESDN or CLCP1, is a 775 amino acid single-pass type I membrane protein containing one CUB domain, one LCCL domain, and one F5/8 type C domain. It has emerged as significant in research due to its diverse roles in cellular processes including regulation of cell growth, angiogenesis, and involvement in cancer development and progression. Research indicates DCBLD2 is highly expressed in testis, heart, skeletal muscle, and vascular smooth muscle cells, making it an important target for studies in both normal physiology and pathological states .

What is the molecular weight discrepancy observed with DCBLD2 in immunoblotting?

While the theoretical molecular weight of human DCBLD2 based on amino acid sequence is approximately 85-89 kDa, it consistently appears at 120-130 kDa on SDS-PAGE. This size discrepancy is attributed to extensive post-translational modifications, particularly glycosylation, as noted in research findings. When designing experiments, researchers should anticipate detecting bands at approximately 120 kDa, and possibly additional bands at 93 kDa and 127 kDa in some tissue samples . This variance highlights the importance of validation studies when working with new antibodies or tissue types.

What are the common synonyms and alternative names for DCBLD2?

When searching literature or antibody databases, researchers should be aware of multiple nomenclatures for this protein:

• DCBLD2 (Discoidin, CUB and LCCL Domain Containing 2) - official gene symbol

• ESDN (Endothelial and smooth muscle cell-derived neuropilin-like protein)

• CLCP1 (CUB, LCCL and coagulation factor V/VIII-homology domains protein 1)

• 1700055P21Rik (mouse ortholog designation)

These alternative designations appear across different research studies and antibody suppliers, making cross-referencing essential when evaluating experimental findings .

How should researchers select the optimal DCBLD2 antibody epitope for their experiments?

Selection of the optimal DCBLD2 antibody should be based on:

• Target domain specificity:

  • N-terminal domain (AA 80-164): Useful for detecting full-length protein

  • CUB domain region (AA 201-300): Often used for structural studies

  • Mid-region (AA 399-416): Common in many commercial antibodies

  • C-terminal region (AA 550-750): Advantageous for membrane-integrated protein detection

• Research application compatibility: Different epitopes perform optimally in specific applications. For example, antibodies targeting AA 201-300 have demonstrated strong performance in IF and IHC applications, while those targeting the C-terminal region (AA 538-567) often show better results in WB and FACS .

• Cross-reactivity requirements: If cross-species reactivity is needed, antibodies targeting the AA 399-416 region show broader reactivity across human, mouse, rat, and hamster samples .

The methodological choice should align with the specific research question and experimental system.

What validation procedures should be employed for DCBLD2 antibodies?

Robust validation of DCBLD2 antibodies should include:

• Positive control selection:

  • Cell lines: HepG2, A549, U2OS, and HCT116 cells demonstrate reliable DCBLD2 expression

  • Tissues: Human liver, brain, heart, testis, and skeletal muscle tissues show high endogenous expression

• Multi-technique validation protocol:

  • Western blot validation: Verify expected molecular weight (120-130 kDa)

  • Knockdown/knockout validation: siRNA or shRNA targeting DCBLD2 should reduce signal intensity

  • Cross-application verification: Confirm consistent staining patterns across WB, IHC, and IF

• Specificity assessment:

  • Peptide competition assay to confirm epitope specificity

  • Multiple antibody comparison (different epitopes) to confirm consistent localization patterns

This methodological approach ensures experimental reproducibility and data reliability across different research applications .

How do different applications affect DCBLD2 antibody dilution requirements?

Based on compiled research data, optimal working dilutions for DCBLD2 antibodies vary significantly by application:

These ranges represent starting points, with sample-dependent optimization required for optimal signal-to-noise ratio. Each new experimental system requires individual titration for optimal results .

What buffer systems optimize DCBLD2 antibody performance in immunohistochemistry?

Research findings indicate DCBLD2 antibody performance in IHC varies significantly with buffer selection:

• Antigen retrieval buffer optimization:

  • Primary recommendation: TE buffer at pH 9.0 has demonstrated superior epitope exposure

  • Alternative option: Citrate buffer at pH 6.0 provides acceptable results for some tissue types

  • Heat-induced epitope retrieval (HIER) outperforms proteolytic methods

• Blocking and incubation conditions:

  • 5% BSA in PBS with 0.1% Tween-20 minimizes background in most tissue samples

  • Overnight incubation at 4°C improves signal specificity compared to shorter incubations

  • Signal amplification systems (e.g., ABC method) significantly improve detection of low-expression samples

• Tissue-specific considerations:

  • Heart tissue: Requires extended blocking (2 hours minimum) to reduce background

  • Cancer tissues: Often benefit from dual peroxidase/protein blocking steps

This methodological approach consistently improves detection specificity across diverse tissue types .

How should researchers interpret multiple band patterns in DCBLD2 Western blots?

The appearance of multiple bands in DCBLD2 Western blots requires careful interpretation:

• Expected band pattern analysis:

  • Primary band: 120-130 kDa (fully glycosylated mature protein)

  • Secondary bands: 93 kDa and 127 kDa (tissue-dependent isoforms or processing variants)

  • Lower molecular weight bands (~85 kDa): May represent non-glycosylated forms

• Validation approach for multiple bands:

  • Glycosidase treatment: PNGase F treatment should shift higher molecular weight bands

  • Denaturation conditions: Varying SDS concentration and heat can affect band patterns

  • Comparison across antibodies targeting different epitopes

• Isoform-specific considerations:

  • Up to 2 different isoforms have been reported for DCBLD2 protein

  • Different tissue types may express variable isoform ratios

This analytical framework helps distinguish between specific signal, isoforms, and non-specific binding .

What are the key considerations for quantitative analysis of DCBLD2 expression?

For accurate quantitative analysis of DCBLD2 expression:

• Reference gene/protein selection:

  • β-actin and GAPDH show variable correlation with DCBLD2 expression

  • Membrane protein controls (e.g., Na+/K+ ATPase) provide better normalization for membrane-localized DCBLD2

• Signal quantification methodology:

  • Integrated density values rather than peak intensity better represent total protein expression

  • For IHC quantification, H-score or Allred scoring systems accounting for both intensity and percentage of positive cells yield more reproducible results

  • For IF analysis, z-stack acquisition improves accuracy of membrane protein quantification

• Expression heterogeneity considerations:

  • Single-cell analysis may be necessary in heterogeneous tissues

  • Microdissection techniques improve quantification in complex tissues

This methodological framework enhances reproducibility and validity of DCBLD2 expression studies .

How can DCBLD2 antibodies be utilized to study its role in cancer progression and drug resistance?

Based on recent research findings, DCBLD2 antibodies can be strategically employed to investigate cancer mechanisms:

• Colorectal cancer research applications:

  • IHC analysis reveals DCBLD2 overexpression correlates with poor prognosis and 5-FU resistance

  • Dual staining with EMT markers (E-cadherin, vimentin) reveals mechanism of DCBLD2-mediated invasion

  • Co-IP experiments demonstrate DCBLD2 interaction with ITGB1, a key factor in Focal adhesion pathway

• Angiogenesis investigation methodology:

  • DCBLD2 and CD31 co-staining identifies regions of active angiogenesis

  • HUVEC cells with silenced DCBLD2 show reduced endothelial characteristics

  • DCBLD2 antibodies can block interaction with VEGFR-2, providing mechanistic insights

• Drug resistance mechanism elucidation:

  • Phospho-specific antibodies can detect DCBLD2 activation status in response to chemotherapy

  • Combined analysis of DCBLD2 and drug transporter expression improves prediction accuracy

These methodological approaches provide mechanistic insights into DCBLD2's role in cancer progression and treatment response .

What are the technical considerations for studying DCBLD2 protein-protein interactions?

For effective analysis of DCBLD2 protein interactions:

• Co-immunoprecipitation optimization:

  • Membrane protein extraction requires specialized buffers containing 1% NP-40 or Triton X-100

  • Cross-linking with DSP (dithiobis(succinimidyl propionate)) improves detection of transient interactions

  • Sequential IP strategy can isolate specific complexes (e.g., DCBLD2-ITGB1)

• Proximity ligation assay (PLA) considerations:

  • Primary antibodies must be raised in different species

  • Fixed cell preparations require careful membrane permeabilization optimization

  • Quantification should include both interaction frequency and subcellular localization analysis

• Advanced techniques for specific interaction studies:

  • TAP-MS (Tandem Affinity Purification-Mass Spectrometry) has successfully identified DCBLD2 binding partners

  • FRET/BRET approaches require specific antibody conjugation and validation

These methodological considerations enhance detection sensitivity and specificity when analyzing DCBLD2's diverse interaction network .

How can phosphorylation-specific DCBLD2 antibodies advance signaling pathway research?

Research into DCBLD2 signaling mechanisms can be enhanced through phospho-specific approaches:

• Key phosphorylation sites for antibody targeting:

  • Tyrosine phosphorylation sites: Critical for interaction with SH2 domain-containing proteins

  • Specific sites in the intracellular domain mediate downstream signaling events

  • Phosphorylation-dependent epitope masking must be considered in experimental design

• Methodological framework for phosphorylation studies:

  • Phosphatase inhibitor cocktails are essential during sample preparation

  • Combined use of general DCBLD2 and phospho-specific antibodies provides relative phosphorylation status

  • Receptor activation studies require careful temporal analysis (15 seconds to 120 minutes)

• Pathway analysis integration:

  • DCBLD2 phosphorylation in relation to Focal adhesion pathway activation

  • EMT signaling pathway connections through phosphorylation-dependent interactions

  • VEGFR-2 cross-talk through phosphorylation cascades

This phospho-specific approach provides crucial insights into the molecular mechanisms of DCBLD2 function in normal and pathological conditions .

How can DCBLD2 antibodies be adapted for multiplexed imaging applications?

Emerging multiplexed imaging approaches for DCBLD2 research include:

• Multi-epitope targeting strategy:

  • Complementary antibodies targeting different DCBLD2 domains enhance detection reliability

  • Different species-derived antibodies enable simultaneous detection with other markers

  • Conjugation with distinct fluorophores permits colocalization studies with interaction partners

• Technical implementation considerations:

  • Sequential immunostaining with antibody stripping between rounds

  • Spectral unmixing to resolve overlapping fluorescence signals

  • Multi-round immunofluorescence with signal elimination between cycles

• Analytical approaches for complex datasets:

  • Machine learning algorithms for pattern recognition in multiplexed images

  • Spatial correlation analysis between DCBLD2 and microenvironmental features

  • Cell type-specific expression quantification in heterogeneous tissues

This methodological framework enables comprehensive spatial analysis of DCBLD2 in complex tissue architectures .

What methodological approaches can elucidate DCBLD2's role in angiogenesis and EMT processes?

Based on recent findings, investigators can implement specific methodologies to study DCBLD2's dual role:

• Angiogenesis research protocol:

  • Endothelial cell tube formation assays with DCBLD2 antibody blocking

  • Co-culture systems with DCBLD2-expressing tumor cells and endothelial cells

  • In vivo Matrigel plug assays comparing DCBLD2 wild-type and knockdown conditions

• EMT process investigation:

  • Sequential sampling during EMT progression with epithelial/mesenchymal marker co-staining

  • Chromatin immunoprecipitation to identify DCBLD2-regulated genes in EMT

  • 3D organoid cultures to visualize EMT spatial dynamics in relation to DCBLD2 expression

• Integrated analytical approach:

  • Correlation analysis between DCBLD2 expression, CD31 (angiogenesis marker), and EMT markers

  • TCGA data mining to identify clinical correlations with combined marker patterns

  • Treatment response prediction based on DCBLD2-associated pathway activation

This comprehensive approach addresses the complementary and potentially synergistic roles of DCBLD2 in tumor progression .

How can researchers optimize DCBLD2 antibodies for therapeutic target validation studies?

For therapeutic target validation involving DCBLD2:

• Antibody functionality assessment:

  • Neutralizing capability evaluation through functional assays

  • Internalization kinetics measurement for potential antibody-drug conjugate applications

  • Epitope mapping to identify functionally critical domains

• In vivo validation methodologies:

  • Xenograft models with antibody treatment regimens

  • Patient-derived organoid testing for response prediction

  • Combination therapy approaches with standard chemotherapeutics

• Predictive biomarker development:

  • Correlation of DCBLD2 expression patterns with treatment outcomes

  • Multi-parameter analysis including phosphorylation status and interaction partners

  • Machine learning algorithms for response prediction based on DCBLD2 and associated pathways