DISP1 Antibody

What is DISP1 and what cellular functions make it relevant for antibody-based research?

DISP1 (dispatched homolog 1) is a 1524 amino acid, 171 kDa transmembrane protein that plays a critical role in the Hedgehog signaling pathway. It functions primarily in the secretion and paracrine distribution of cholesterol-modified Hedgehog ligands during embryonic development. The protein is encoded by the DISP1 gene (Gene ID: 84976) and shares homology with its Drosophila counterpart .

Research utilizing DISP1 antibodies has significant relevance in developmental biology, cancer research, and congenital disorder investigations. The protein's large molecular weight (observed at 150-171 kDa in experimental conditions) and transmembrane nature present specific challenges for antibody-based detection that researchers must account for in experimental design .

What are the validated applications for DISP1 antibody in research settings?

DISP1 antibody (such as the 12041-1-AP clone) has been validated for multiple research applications with human samples. The primary validated applications include:

It is essential to note that the optimal dilution is highly dependent on sample type and experimental conditions. Researchers should perform titration experiments to determine optimal antibody concentration for their specific experimental setup .

What are the optimal protocols for DISP1 antibody in Western Blot applications?

For Western Blot applications using DISP1 antibody, researchers should follow these methodological guidelines:

• Sample preparation: Prepare protein lysates from target cells (HEK-293 and HeLa cells have shown positive results) using standard lysis buffers containing protease inhibitors.

• Protein separation: Due to DISP1's high molecular weight (150-171 kDa), use a low percentage (6-8%) SDS-PAGE gel or gradient gel to ensure proper resolution.

• Transfer: Employ wet transfer methods using low methanol buffer to facilitate efficient transfer of high molecular weight proteins.

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute DISP1 antibody at 1:500-1:3000 in blocking buffer and incubate overnight at 4°C.

• Detection: Use appropriate secondary antibody (anti-rabbit IgG) and visualization system.

Researchers should anticipate bands in the 150-171 kDa range, which corresponds to the observed molecular weight of the DISP1 protein .

What antigen retrieval methods are recommended for DISP1 immunohistochemistry?

For optimal immunohistochemical detection of DISP1 in tissue samples, antigen retrieval is a critical step. The recommended protocols include:

• Primary recommendation: TE buffer pH 9.0 for heat-induced epitope retrieval (HIER).

• Alternative method: Citrate buffer pH 6.0 can be used if the primary method yields suboptimal results.

When performing IHC with DISP1 antibody:

• Use a dilution range of 1:50-1:500, with optimization recommended for each tissue type

• Human kidney tissue has been confirmed as a positive control

• Incubate sections with primary antibody overnight at 4°C to maximize specific binding

• Use appropriate secondary detection systems compatible with rabbit IgG

What are the proper storage and handling conditions for DISP1 antibody?

To maintain optimal reactivity and specificity, DISP1 antibody requires specific storage and handling conditions:

The antibody is supplied in liquid form and has undergone antigen affinity purification to ensure specificity. Following these handling guidelines will help maintain antibody performance throughout the research project timeline .

How can researchers validate DISP1 antibody specificity for their experimental system?

Validating antibody specificity is crucial for ensuring reliable research results. For DISP1 antibody validation, researchers should implement a multi-faceted approach:

• Positive and negative controls:

  • Use cell lines with known DISP1 expression (HEK-293, HeLa) as positive controls

  • Include DISP1 knockout or knockdown samples as negative controls

  • Consider tissue specificity (kidney tissue has confirmed positivity)

• Molecular weight verification:

  • Confirm band appearance at the expected molecular weight (150-171 kDa)

  • Be aware that post-translational modifications may cause slight variations in observed weight

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide (DISP1 fusion protein Ag2670)

  • Specific signal should be significantly reduced or eliminated

• Orthogonal method validation:

  • Confirm findings using alternative detection methods (e.g., mass spectrometry)

  • Compare results with alternative antibody clones if available

• Genetic manipulation validation:

  • Overexpress DISP1 to confirm increased signal intensity

  • Use RNA interference to demonstrate reduced signal proportional to knockdown efficiency

What are the critical considerations for antibody-antigen interaction analysis in DISP1 research?

When investigating DISP1 antibody-antigen interactions, researchers should consider several critical factors:

• Structural complexity: DISP1 is a multi-pass transmembrane protein, which presents unique challenges for epitope accessibility and antibody binding. The three-dimensional conformation significantly impacts antibody recognition.

• Epitope mapping considerations:

  • The 12041-1-AP antibody targets a specific fusion protein region (Ag2670)

  • Native protein folding may affect epitope accessibility differently than in denatured conditions

• Cross-reactivity assessment:

  • Verify specificity against related proteins in the dispatched family

  • Consider potential non-specific interactions with membrane proteins of similar structural motifs

• Quantitative binding analysis:

  • Assess antibody affinity using surface plasmon resonance or bio-layer interferometry

  • Determine binding kinetics to understand association/dissociation rates

• Model quality evaluation:

  • When modeling antibody-antigen interactions, apply rigorous statistical analysis

  • Consider both DOCKQ score and bRD p-value for comprehensive evaluation

  • Models with bRD p-value below 0.01 are classified as "significant"

These considerations are essential for proper interpretation of results and experimental reproducibility when studying DISP1 using antibody-based methods.

How can researchers troubleshoot non-specific binding issues with DISP1 antibody?

Non-specific binding is a common challenge in DISP1 antibody applications that can compromise data interpretation. Here's a methodological approach to troubleshooting:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time or concentration if background is high

  • Consider specialized blocking reagents for membrane proteins

• Adjust antibody concentration:

  • Perform thorough titration experiments (starting with recommended dilutions of 1:500-1:3000 for WB)

  • Balance signal strength with background minimization

  • Consider longer incubation times with more dilute antibody

• Modify washing procedures:

  • Increase number of washes or washing buffer stringency

  • Add low concentrations of detergents to reduce hydrophobic interactions

  • Implement longer washing steps to remove weakly bound antibody

• Sample preparation refinement:

  • Ensure complete protein denaturation for WB applications

  • For native applications, verify membrane protein solubilization is appropriate

  • Remove lipid content that may cause non-specific hydrophobic interactions

• Cross-validation strategies:

  • Compare results between different detection methods

  • Utilize negative controls lacking DISP1 expression

  • Implement peptide competition controls

What advanced methodology enables high-throughput screening with DISP1 antibody?

For researchers conducting large-scale or high-throughput studies involving DISP1 antibody, several advanced methodological approaches can be implemented:

• Protein array technology:

  • High density-nucleic acid programmable protein arrays (HD-NAPPA) offer superior signal-to-background ratios compared to standard ELISA

  • Enable simultaneous analysis of multiple protein-antibody interactions

  • Allow for comparative studies across different experimental conditions

• Automated Western Blot systems:

  • Implement capillary-based automated Western technologies for higher reproducibility

  • Utilize microfluidic platforms for reduced sample consumption

  • Apply computational image analysis for standardized quantification

• Multiplex immunoassays:

  • Develop bead-based multiplex assays for simultaneous detection of DISP1 and related proteins

  • Implement appropriate controls to account for potential cross-reactivity

  • Validate assay specificity through spike-recovery experiments

• High-content imaging:

  • Apply automated microscopy for IHC/ICC applications

  • Implement machine learning algorithms for pattern recognition and quantification

  • Develop standardized analysis pipelines for consistent data interpretation

• Quality control considerations:

  • Establish intra- and inter-array correlation coefficients (target >0.90)

  • Implement standard reference samples across experimental batches

  • Develop robust normalization strategies for cross-platform comparisons

How can researchers integrate DISP1 antibody data with other molecular profiling approaches?

Integrating DISP1 antibody-derived data with complementary molecular profiling approaches provides a more comprehensive understanding of biological systems. Researchers should consider these methodological strategies:

• Multi-omics integration framework:

  • Correlate DISP1 protein expression (antibody-based) with transcriptomic data

  • Integrate proteomic and interactome data to place DISP1 in functional networks

  • Apply computational algorithms to identify regulatory relationships

• Spatial analysis integration:

  • Combine IHC data with spatial transcriptomics

  • Correlate antibody staining patterns with tissue microenvironment features

  • Implement digital pathology tools for quantitative spatial analysis

• Temporal profiling strategies:

  • Design time-course experiments measuring DISP1 expression during development or disease progression

  • Correlate protein-level changes with transcriptional dynamics

  • Apply mathematical modeling to infer causal relationships

• Functional validation approaches:

  • Complement antibody detection with CRISPR-based functional studies

  • Verify protein-protein interactions using proximity ligation assays

  • Combine antibody detection with functional readouts of Hedgehog pathway activity

• Data harmonization techniques:

  • Apply appropriate normalization methods across platforms

  • Implement batch correction algorithms when combining datasets

  • Use standard reference samples for cross-experiment calibration

This integrated approach enables researchers to place DISP1 antibody findings within a broader biological context and generate more robust hypotheses for future investigation.

What are the emerging applications for DISP1 antibody in current research?

DISP1 antibody applications continue to evolve as our understanding of Hedgehog signaling pathways deepens. Current research frontiers include:

• Developmental biology: Investigating DISP1's role in embryonic patterning and tissue specification through detailed spatiotemporal expression analysis.

• Cancer research: Exploring DISP1's contribution to aberrant Hedgehog signaling in various malignancies, with potential therapeutic implications.

• Stem cell biology: Examining DISP1's function in maintaining stemness and directing differentiation through paracrine signaling mechanisms.

• Precision medicine: Correlating DISP1 expression patterns with patient outcomes and treatment responses in Hedgehog-dependent pathologies.

• Drug development: Using DISP1 antibodies to screen for compounds that modulate Hedgehog ligand secretion as a novel therapeutic approach.

These applications highlight the continued importance of high-quality, well-validated DISP1 antibodies in advancing our understanding of fundamental biological processes and disease mechanisms .

What quality control metrics should researchers apply when evaluating DISP1 antibody performance?

To ensure reliable and reproducible results, researchers should implement rigorous quality control metrics when using DISP1 antibody:

• Batch-to-batch consistency:

  • Validate each new antibody lot against previous lots

  • Document and compare key performance parameters across experiments

  • Maintain reference samples for standardized comparisons

• Sensitivity assessments:

  • Determine limit of detection in relevant experimental systems

  • Quantify signal-to-noise ratio under standardized conditions

  • Assess linearity of response across a range of antigen concentrations

• Reproducibility metrics:

  • Calculate intra- and inter-assay coefficients of variation

  • Implement positive controls with known DISP1 expression levels

  • Establish acceptable performance ranges for key experimental readouts

• Specificity validation:

  • Regularly perform peptide competition assays

  • Compare results with orthogonal detection methods

  • Validate against genetic knockout or knockdown systems when available

• Application-specific quality controls:

  • For WB: verify molecular weight and band pattern consistency

  • For IHC: assess staining pattern against established cellular localization

  • For multiplex applications: evaluate for potential cross-reactivity