DAG1 Antibody, Biotin conjugated

What is the molecular target of DAG1 Antibody, Biotin conjugated?

DAG1 Antibody targets dystroglycan 1, a protein involved in multiple cellular processes including laminin and basement membrane assembly, sarcolemmal stability, cell survival, peripheral nerve myelination, nodal structure, cell migration, and epithelial polarization. The protein undergoes post-translational cleavage into two functional subunits: alpha-dystroglycan (an extracellular peripheral glycoprotein) and beta-dystroglycan (a transmembrane protein) .

The specific antibody discussed here recognizes a peptide sequence from the human dystroglycan protein (amino acids 726-744), which is located in the C-terminal region . This region is part of the beta-dystroglycan domain that plays important roles in connecting the extracellular matrix to the cytoskeleton.

What are the technical specifications of this antibody?

The DAG1 Antibody, Biotin conjugated has the following specifications:

Source: Combined data from multiple technical datasheets

What is the advantage of using a biotin-conjugated antibody for DAG1 detection?

The biotin conjugation provides significant advantages for DAG1 detection in several experimental contexts:

• Signal Amplification: Biotin forms a strong non-covalent interaction with avidin/streptavidin (Kd ≈ 10^-15 M), one of the strongest non-covalent interactions in nature. This allows for signal amplification as multiple detection molecules (e.g., enzyme-linked streptavidin) can bind to each biotin molecule.

• Versatility in Detection Systems: The biotin tag enables flexibility in choosing secondary detection reagents. Researchers can use streptavidin conjugated to various reporter molecules (HRP, fluorophores, gold particles) depending on the experimental needs.

• Reduced Background: The biotin-streptavidin system often provides cleaner results with less non-specific binding compared to traditional secondary antibody approaches, particularly important when working with complex tissue samples where DAG1 may be expressed at varying levels .

• Multi-step Detection Protocols: Allows for layered detection strategies, which can be particularly useful when studying DAG1's interactions with other proteins in complex cellular environments.

What are the optimized protocols for using DAG1 Antibody, Biotin conjugated in ELISA?

Based on compiled research methodologies, here is an optimized ELISA protocol for DAG1 Antibody, Biotin conjugated:

• Coating: Coat 96-well microplate with DAG1 antigen or sample containing DAG1 protein (typically 1-10 μg/ml in carbonate buffer pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 5% non-fat dry milk or 3% BSA in PBS for 1-2 hours at room temperature.

• Primary Antibody: Dilute DAG1 Antibody, Biotin conjugated at 1:500-1:2000 in blocking buffer and incubate for 1-2 hours at room temperature.

• Detection: Add streptavidin-HRP (typically 1:5000-1:10000) and incubate for 30-60 minutes at room temperature.

• Substrate: Add TMB substrate and monitor color development.

• Stop Reaction: Add stop solution (usually 2N H₂SO₄) and read absorbance at 450 nm.

For sandwich ELISA, coat the plate with a capture antibody that recognizes a different epitope of DAG1 than the biotin-conjugated antibody to avoid epitope competition .

Can this antibody be adapted for Western Blot applications despite being optimized for ELISA?

While this specific biotin-conjugated antibody is primarily recommended for ELISA, adapting it for Western Blot may be possible with the following considerations:

• Denaturation Concerns: The epitope (AA 726-744) may be affected by SDS-PAGE denaturation. Related DAG1 antibodies have successfully detected bands at approximately 120-150 kDa (alpha-dystroglycan) and approximately 43 kDa (beta-dystroglycan) .

• Protocol Adaptation:

  • Use a higher concentration (1:200-1:500) than in ELISA

  • Perform transfer to PVDF membrane (preferred over nitrocellulose for glycoproteins like DAG1)

  • Block with 5% non-fat dry milk in TBST

  • Incubate with the biotin-conjugated antibody overnight at 4°C

  • Detect using streptavidin-HRP (1:5000-1:10000)

  • Visualize with enhanced chemiluminescence

• Controls: Include known positive controls such as human skeletal muscle or heart tissue, which show strong DAG1 expression .

• Expected Banding Pattern: Alpha-dystroglycan typically appears as a diffuse band at 120-150 kDa due to extensive glycosylation, while beta-dystroglycan appears at approximately 43 kDa .

What are the recommended antibody combinations for studying DAG1 glycosylation status?

DAG1 glycosylation is critical for its function, particularly for alpha-dystroglycan's ability to bind extracellular matrix components. Research suggests using the following antibody combinations:

• For Detecting Core Protein:

  • Use antibodies targeting the C-terminal region (like this biotin-conjugated antibody) to detect total DAG1 expression regardless of glycosylation status.

• For Detecting Functional Glycosylation:

  • Combine with IIH6C4 clone antibodies (like ab234587) , which specifically recognize the functionally glycosylated form of alpha-dystroglycan.

• Dual Labeling Approach:

  • In immunofluorescence experiments, co-stain with the biotin-conjugated DAG1 antibody (detected with streptavidin-fluorophore) and IIH6C4 antibody (detected with a different fluorophore).

  • Ratio of signals provides information about the proportion of properly glycosylated DAG1.

This approach is particularly useful for research on muscular dystrophies, where altered glycosylation of alpha-dystroglycan is often observed .

How can I address weak or absent signals when using DAG1 Antibody, Biotin conjugated?

When experiencing weak or absent signals, consider these methodological solutions:

• Antibody Concentration:

  • Increase antibody concentration (try a titration series: 1:250, 1:500, 1:1000)

  • Extended incubation time (overnight at 4°C instead of 1-2 hours at room temperature)

• Sample Preparation:

  • Ensure proper extraction of membrane proteins (DAG1 is a transmembrane protein)

  • Use specialized lysis buffers containing 1% Triton X-100 or NP-40

  • Add protease inhibitors to prevent degradation

• Antigen Retrieval (for tissue sections):

  • For paraffin sections, try heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • For frozen sections, fix with 4% paraformaldehyde for optimal epitope preservation

• Signal Amplification:

  • Use tyramide signal amplification (TSA) with the biotin-streptavidin system

  • Consider using anti-biotin antibodies conjugated to HRP as an alternative detection method

• Storage and Handling:

  • Avoid repeated freeze-thaw cycles which can degrade antibody activity

  • Store as recommended at -20°C or -80°C in small aliquots

Why might I observe inconsistent results when comparing DAG1 antibody data across different species?

Inconsistencies across species may arise from several factors:

• Sequence Homology Variations:

  • The human DAG1 immunogen (AA 726-744) shows variable conservation across species

  • While the antibody is primarily validated for human samples, cross-reactivity with mouse and rat samples may vary based on epitope conservation

• Species-Specific Glycosylation Patterns:

  • Alpha-dystroglycan glycosylation varies significantly across species

  • This affects antibody accessibility to the core protein and may cause inconsistent results

• Expression Level Differences:

  • DAG1 expression levels vary across tissues and species

  • In zebrafish models (like the patchytail mutant), DAG1 mutations can lead to complete absence of detectable protein

• Technical Recommendations:

  • Always include species-appropriate positive controls

  • Consider using multiple antibodies targeting different DAG1 epitopes

  • Validate antibody reactivity for each new species before conducting comprehensive experiments

What are the advanced applications of DAG1 Antibody, Biotin conjugated in studying muscular dystrophies?

This antibody can be instrumental in several sophisticated research applications:

• Diagnostic Screening:

  • Can be used in multiplex ELISA systems to detect DAG1 abnormalities in patient samples

  • Particularly valuable for diagnosing dystroglycanopathies, where DAG1 glycosylation is compromised

• Therapeutic Monitoring:

  • In gene therapy or small molecule interventions targeting DAG1 glycosylation

  • Quantitative ELISA using this antibody can measure restoration of DAG1 expression

• Mechanistic Studies:

  • Investigation of DAG1 processing in different muscular dystrophy models

  • Combined with mass spectrometry to analyze post-translational modifications

• High-Throughput Screening:

  • Adaptation for 384-well plate format ELISA for screening compounds that may affect DAG1 expression or processing

  • Biotin conjugation enables compatibility with automated detection systems

• In vivo Imaging:

  • When combined with appropriate in vivo imaging techniques, can be used to track DAG1 distribution in animal models

  • Particularly useful in zebrafish models where real-time imaging is possible

How does the epitope location (AA 726-744) relate to DAG1 functional domains?

The amino acid sequence 726-744 targeted by this antibody is functionally significant:

• Domain Location:

  • This sequence is located in the C-terminal region of beta-dystroglycan

  • Part of the cytoplasmic domain that interacts with the cytoskeleton

• Functional Significance:

  • Contains binding sites for dystrophin and utrophin

  • Critical for signal transduction from the extracellular matrix to the cytoskeleton

  • Contains potential phosphorylation sites that regulate protein-protein interactions

• Conservation:

  • This region shows high conservation across vertebrates, indicating its functional importance

  • Mutations or modifications in this region can disrupt the dystrophin-glycoprotein complex

• Research Applications:

  • Antibodies targeting this region are valuable for studying the membrane anchoring and signaling functions of DAG1

  • Can be used to investigate how DAG1 processing affects its interactions with cytoskeletal proteins

How can DAG1 Antibody, Biotin conjugated be used to investigate disease mechanisms in dystroglycanopathies?

Dystroglycanopathies, a subset of muscular dystrophies, are characterized by abnormal glycosylation of alpha-dystroglycan. This antibody can be utilized to:

• Distinguish Between Expression and Glycosylation Defects:

  • Total DAG1 protein can be detected regardless of glycosylation status

  • Comparing results with glycosylation-specific antibodies helps distinguish between expression defects and glycosylation abnormalities

• Evaluate Therapeutic Interventions:

  • Monitor restoration of DAG1 expression following gene therapy

  • Assess the effect of compounds targeting glycosylation enzymes

• Analyze Tissue-Specific Manifestations:

  • Compare DAG1 expression across different affected tissues (muscle, brain, retina)

  • Correlate with clinical phenotypes to understand tissue-specific pathology

• Molecular Pathway Analysis:

  • Combined with antibodies against glycosylation enzymes (POMT1, POMT2, POMGnT1, etc.)

  • Use in co-immunoprecipitation studies to identify novel interacting partners

• Zebrafish Models:

  • The patchytail zebrafish mutant provides a valuable model for dystroglycanopathies

  • This antibody can help characterize DAG1 expression patterns and defects in this model

What are the technical considerations when comparing multiple dystroglycan antibodies in the same experiment?

When using multiple DAG1 antibodies in a single experiment:

• Epitope Mapping and Competition:

  • Antibodies targeting overlapping epitopes may compete and interfere with each other

  • This biotin-conjugated antibody (AA 726-744) targets the C-terminal region and should be paired with antibodies targeting distant epitopes

• Host Species Considerations:

  • This rabbit-derived antibody should ideally be paired with antibodies from different host species (mouse, goat) to avoid cross-reactivity of secondary antibodies

• Detection Strategy:

  • The biotin conjugation provides a distinct advantage for multiplexing

  • Can be detected with streptavidin conjugated to a spectrally distinct fluorophore from secondary antibodies

• Sequential Staining Protocol:

  • For multiple rabbit antibodies, consider sequential staining with complete blocking between antibodies

  • Apply the biotin-conjugated antibody first, detect with streptavidin-conjugate, then block biotin/streptavidin with avidin/biotin blocking kit before applying the next antibody

• Validation Controls:

  • Include both positive (tissues known to express DAG1) and negative controls (DAG1 knockout samples)

How can DAG1 Antibody, Biotin conjugated contribute to emerging research on DAG1's role in non-muscle tissues?

While DAG1 is well-studied in muscular contexts, its functions in other tissues are increasingly recognized:

• Neural Development and Function:

  • This antibody can be used to study DAG1's role in peripheral nerve myelination

  • Applicable in co-localization studies with neuronal markers in primary hippocampal neuron cultures

• Cancer Research:

  • DAG1 has emerging roles in cell migration and cancer progression

  • The antibody can be used to assess DAG1 expression in tumor samples and cancer cell lines (such as U87-MG)

• Epithelial Cell Biology:

  • DAG1 contributes to epithelial polarization and basement membrane assembly

  • This antibody can be used in pancreatic and other epithelial tissues to study these processes

• Infectious Disease Research:

  • Alpha-dystroglycan serves as a receptor for certain pathogens including Lassa virus and Mycobacterium leprae

  • The antibody can help study receptor-pathogen interactions in infection models

• Methodological Approaches:

  • Combination with tissue-specific markers for co-localization studies

  • Application in patient-derived organoids to study tissue-specific DAG1 functions

What are the emerging technical innovations in DAG1 antibody applications?

Recent technical advances are expanding the utility of DAG1 antibodies like this biotin-conjugated variant:

• Single-Cell Analysis:

  • Adaptation for mass cytometry (CyTOF) using metal-conjugated streptavidin

  • Application in single-cell Western blotting to detect DAG1 variants at the single-cell level

• Super-Resolution Microscopy:

  • Utilization with techniques like STORM or PALM for nanoscale localization

  • Biotin-streptavidin detection systems are particularly valuable for these applications due to their high signal-to-noise ratio

• Tissue Clearing Techniques:

  • Compatibility with CLARITY, iDISCO, or other tissue clearing methods

  • Enables 3D visualization of DAG1 distribution in intact tissues

• Proximity Ligation Assays (PLA):

  • Combining this antibody with antibodies against potential interacting partners

  • Allows detection of protein-protein interactions in situ with high sensitivity

• Automation and High-Throughput Applications:

  • Integration into automated immunoassay platforms

  • Development of DAG1 antibody arrays for comprehensive profiling

How can researchers optimize experimental design when studying DAG1 in complex genetic disorders?

When investigating DAG1 in genetic disorders, consider these advanced experimental design principles:

• Genetic Background Considerations:

  • DAG1 function may be modified by genetic background

  • Include appropriate genetic controls when using model organisms

  • Consider using isogenic cell lines (created using CRISPR/Cas9) that differ only in DAG1 sequence

• Tissue-Specific Conditional Models:

  • DAG1 knockout is embryonic lethal in many organisms

  • Use tissue-specific or inducible knockout models to study function in specific contexts

  • This antibody can help validate the efficiency of conditional knockout

• Multi-omics Integration:

  • Combine antibody-based detection with transcriptomics and proteomics

  • Correlate DAG1 protein levels with glycosylation enzyme expression

  • Integrate with patient genomic data to establish genotype-phenotype correlations

• Longitudinal Studies:

  • Track DAG1 expression during disease progression

  • Design time-course experiments to capture dynamic changes

  • Use the antibody in repeated sampling from the same experimental subjects

• Translational Research Pipeline:

  • Bridge basic science findings to clinical applications

  • Establish standardized protocols for DAG1 assessment in patient samples

  • Develop biomarker panels that include DAG1 measurements for disease monitoring