dlg1l Antibody

What is DLG1 and why is it important in research?

DLG1 (Discs Large Homolog 1) is a member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins. It plays critical roles in cell-cell adhesion, signal transduction, and cellular polarity. In C. elegans, DLG-1 is primarily associated with adherens junctions, similar to its homologs in other species . The protein contains multiple PDZ domains that facilitate protein-protein interactions, making it an important subject for studies in developmental biology, neuroscience, and cancer research. DLG1 antibodies are essential tools for investigating these functions across various experimental systems.

What types of DLG1 antibodies are available for research?

Researchers can access several types of DLG1 antibodies with various specificities:

• Polyclonal antibodies: These recognize multiple epitopes on the DLG1 protein, such as rabbit IgG polyclonal antibodies targeting amino acids 1-165 of human DLG1 .

• Monoclonal antibodies: These target specific epitopes, like the mouse monoclonal (clone DLG1/5E2) that recognizes amino acids 204-593 (PDZ domains 1-3) of C. elegans DLG-1 .

• Domain-specific antibodies: Various antibodies target specific regions, including those recognizing the N-terminal region (AA 1-165), PDZ domains (AA 204-593), and C-terminal regions (AA 801-904) .

What species reactivity do commercial DLG1 antibodies offer?

DLG1 antibodies exhibit different species reactivities depending on their design and the conservation of target epitopes:

• Human, mouse, and rat reactivity: Many commercially available antibodies, such as the rabbit polyclonal against AA 1-165, show cross-reactivity across these mammalian species .

• C. elegans specificity: Specialized antibodies like the DLG1 monoclonal from DSHB are designed for C. elegans research with confirmed species reactivity .

• No cross-reactivity: Some antibodies are highly specific and show no cross-reactivity with other proteins, which is valuable for distinguishing between closely related family members .

What are the recommended applications for DLG1 antibodies?

DLG1 antibodies can be used in multiple experimental techniques:

• Western blotting: Both polyclonal and monoclonal DLG1 antibodies perform well in WB applications, detecting the protein at its predicted molecular weight of approximately 100-107 kDa .

• Immunohistochemistry: Paraffin-embedded section IHC (IHC-P) is supported by polyclonal antibodies like ABIN3043562 .

• Immunofluorescence: The monoclonal antibody from DSHB works particularly well for IF applications, especially for C. elegans whole mounts .

• Immunocytochemistry: Some DLG1 antibodies are validated for ICC applications, allowing subcellular localization studies .

How should I optimize DLG1 antibody dilutions for different applications?

Optimal dilutions vary by application and antibody format:

• For immunohistochemistry, immunofluorescence, and immunocytochemistry:

  • Mouse monoclonal antibodies: Initial testing at 2-5 μg/ml is recommended

  • Rabbit polyclonal antibodies: Start with 0.2-0.5 μg/ml due to typically higher affinity

• For Western blotting:

  • Mouse monoclonal antibodies: 0.2-0.5 μg/ml is typically effective

  • Rabbit polyclonal antibodies: 20-50 ng/ml is often sufficient

Always perform a dilution series to determine optimal concentration for your specific experimental system, as antibody performance can vary across tissue types and fixation methods.

What controls should be used when working with DLG1 antibodies?

Proper controls are essential for validating DLG1 antibody specificity:

• Positive control: Include samples known to express DLG1 (e.g., epithelial tissues for mammalian studies, whole C. elegans lysates for nematode studies)

• Negative control: Samples lacking DLG1 expression or tissues from knockout models

• Isotype control: Use matched isotype antibodies (e.g., rabbit IgG for polyclonal, mouse IgG1 for monoclonal) to identify non-specific binding

• Blocking peptide control: Pre-incubate the antibody with the immunizing peptide to confirm specificity

• Secondary antibody control: Omit primary antibody to detect non-specific binding of secondary antibodies

How should DLG1 antibodies be stored and handled?

Proper storage and handling are critical for maintaining antibody functionality:

• Short-term storage: For immediate use (within two weeks), store at 4°C

• Long-term storage: Divide into small aliquots (≥20 μl) and store at -20°C or -80°C

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces antibody activity

• Cryoprotection: For concentrate products, consider adding an equal volume of glycerol before freezing

• Working dilutions: Prepare fresh working dilutions on the day of use

• Sterile conditions: Use sterile techniques when handling antibody solutions to prevent contamination

What are the differences between polyclonal and monoclonal DLG1 antibodies?

Understanding these differences helps in selecting the appropriate reagent:

How do I troubleshoot non-specific binding with DLG1 antibodies?

Non-specific binding can be addressed through several approaches:

• Increase blocking: Use 5% BSA or 5% milk in TBS-T for Western blots; extend blocking time to 2 hours

• Optimize antibody concentration: Titrate to determine minimal effective concentration

• Increase washing: Add additional wash steps with higher detergent concentration

• Pre-adsorption: Pre-incubate antibody with non-target tissue lysate to remove cross-reactive antibodies

• Change blocking agent: If BSA doesn't work effectively, try normal serum from the secondary antibody host species

• Adjust incubation time/temperature: Shorter incubation at room temperature may reduce non-specific binding

• Pretreat with hydrogen peroxide: For IHC applications, this can reduce endogenous peroxidase activity

How can I verify the specificity of my DLG1 antibody?

Verification of specificity is crucial for confident interpretation of results:

• Knockdown validation: Compare staining patterns between wild-type and DLG1 knockdown/knockout samples

• Epitope mapping: Verify that the detected band/signal corresponds to the expected molecular weight (approximately 107 kDa for C. elegans DLG-1)

• Mass spectrometry: Immunoprecipitate the target protein and confirm identity via mass spectrometry

• Recombinant protein controls: Use purified recombinant DLG1 protein as a positive control

• Multiple antibody approach: Use antibodies targeting different epitopes to confirm results

• RNA-protein correlation: Compare protein expression patterns with RNA expression data

What approaches can help distinguish between closely related DLG family members?

Distinguishing between similar proteins requires carefully designed experiments:

• Epitope selection: Choose antibodies targeting less-conserved regions between family members

• Absorption controls: Pre-absorb antibodies with recombinant proteins of related family members

• Immunodepletion: Sequentially deplete lysates with antibodies against related proteins

• High-resolution imaging: Use super-resolution microscopy to detect differences in subcellular localization

• Co-localization studies: Examine co-localization patterns with known interaction partners specific to each family member

• Isoform-specific PCR: Correlate protein detection with isoform-specific mRNA expression

How do computational approaches enhance antibody specificity determination?

Recent advances in computational biology offer new tools for antibody characterization:

• Biophysics-informed models: Machine learning approaches can identify distinct binding modes associated with specific ligands, enabling prediction and generation of antibody variants with desired specificity profiles

• High-throughput sequencing analysis: Integration of sequencing data with experimental selection can disentangle binding modes, even for chemically similar ligands

• Specificity prediction: Computational models can predict cross-reactivity and specificity beyond what is directly observed in experiments

• Binding mode identification: Statistical analysis can associate sequence patterns with distinct binding interactions, helping identify determinants of specificity

How are high-throughput sequencing and machine learning advancing antibody development?

These technologies are revolutionizing antibody research:

• Beyond observed sequences: Computational approaches can now make predictions beyond experimentally observed sequences, enabling the design of novel antibodies with desired properties

• Multiple property inference: Modern methods can infer multiple physical properties simultaneously, including those not directly measured in selection experiments

• Specificity profile design: Computational tools can design antibodies with customized specificity profiles, either highly specific for a particular target or with controlled cross-specificity for multiple targets

• Counter-selection efficiency: Computational approaches can achieve counter-selection (elimination of off-target binding) more efficiently than experimental methods alone

What are the latest approaches for designing antibodies with customized specificity profiles?

Cutting-edge methods include:

• Phage display with computational analysis: Combining selection experiments with downstream computational analysis provides enhanced control over specificity profiles

• Biophysical modeling: Incorporating biophysical constraints into models offers quantitative insights and improved design capabilities

• Binding mode identification: Associating sequence patterns with distinct binding modes enables the design of antibodies with tailored specificity

• Selection against multiple ligands: Training models on selections against various ligand combinations helps disentangle binding modes associated with specific targets

• Variant prediction: Computational models can propose novel antibody sequences not present in the original library that exhibit desired specificity profiles