DAPP1 Antibody, FITC conjugated

What is DAPP1 and why is it important in immunological research?

DAPP1 (Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides) is a 280 amino acid protein that contains a putative myristoylation site at its N-terminus, followed by an Src homology (SH2) domain and a pleckstrin homology (PH) domain at its C-terminus . This protein, also known as BAM32 (B-cell Adapter Molecule of 32 kDa), functions as a key adaptor protein in B-cell signaling pathways.

DAPP1 exhibits high-affinity interactions with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂), but not with other phospholipids . Its dual-binding capability to both tyrosine-phosphorylated proteins (via the SH2 domain) and 3-phosphoinositides (via the PH domain) makes it a critical regulator in B-cell antigen receptor (BCR) signaling downstream of phosphoinositide 3-kinase (PI3K) .

What is the optimal FITC conjugation protocol for DAPP1 antibodies?

For effective FITC conjugation to DAPP1 antibodies, follow this methodological approach:

• Buffer Preparation: Use 0.1M sodium carbonate buffer at pH 9.0. Avoid stored buffer older than one week as pH fluctuations can occur .

• Antibody Preparation: Ensure the DAPP1 antibody is pure and free of contamination. Dissolve at 2 mg/ml in the carbonate buffer. Avoid buffers containing amines or azides that may compete with the labeling reaction .

• FITC Solution: Prepare fresh 1 mg/ml FITC in anhydrous DMSO immediately before use. Never use stored FITC solutions .

• Conjugation Reaction:

  • Add 50 μl of FITC solution per ml of antibody solution in 5 μl intervals while gently stirring

  • Incubate in the dark at 4°C for 8 hours

  • The isothiocyanate group of FITC reacts with primary amines on the antibody, forming stable thiourea bonds

• Purification: Remove unconjugated FITC through dialysis or gel filtration. For optimal results, separate different F/P ratio populations through gradient DEAE Sephadex chromatography .

What applications are DAPP1-FITC conjugated antibodies most suitable for?

DAPP1-FITC conjugated antibodies are particularly valuable in the following research applications:

*Exact dilutions should be experimentally determined for each specific antibody and application.

The FITC conjugation enables direct fluorescent detection without secondary antibodies, streamlining experimental workflows while maintaining specificity for DAPP1 detection .

What controls should I include when using DAPP1-FITC antibodies?

When designing experiments with DAPP1-FITC antibodies, include these essential controls:

• Isotype Control: Use a FITC-conjugated antibody of the same isotype (e.g., IgG for most DAPP1 antibodies) but with irrelevant specificity to assess non-specific binding .

• Blocking Control: Pre-incubate the DAPP1-FITC antibody with the immunizing peptide to confirm specificity, as demonstrated in validation studies showing complete signal blocking .

• Negative Cell/Tissue Control: Include samples known not to express DAPP1 or samples where DAPP1 has been knocked down/out.

• Positive Control: Use cell lines with confirmed DAPP1 expression (e.g., Ramos or IM-9 cells) as demonstrated in validation studies .

• Autofluorescence Control: Include unstained samples to account for natural cellular fluorescence in the FITC spectrum.

How should DAPP1-FITC conjugated antibodies be stored for maximum stability?

For optimal stability and performance of DAPP1-FITC conjugated antibodies:

• Storage Temperature: Store aliquoted antibody at -20°C for long-term storage. Avoid repeated freeze-thaw cycles that can degrade both the antibody and the fluorophore .

• Short-term Storage: For ongoing experiments, store at 4°C for up to one week .

• Protection from Light: FITC is photosensitive; always store in amber vials or wrapped in aluminum foil to prevent photobleaching .

• Aliquoting: Upon receipt, divide into small single-use aliquots to minimize freeze-thaw cycles.

• Buffer Considerations: Most commercial preparations contain stabilizers, but avoid buffers with sodium azide for applications sensitive to respiratory chain inhibition .

How can DAPP1-FITC antibodies be used to investigate pH-dependent cellular microenvironments?

DAPP1-FITC antibodies can be valuable tools for studying pH-dependent cellular processes, particularly in tumor microenvironments where local acidity affects molecular interactions:

• pH-Dependent Fluorescence: FITC fluorescence intensity is inherently pH-sensitive, decreasing at lower pH. This property can be harnessed as an internal pH indicator when using DAPP1-FITC antibodies in environments with varying acidity .

• Experimental Design:

  • Calibrate fluorescence intensity against standard pH buffers

  • Use ratiometric imaging with a pH-insensitive fluorophore as reference

  • Compare DAPP1-FITC binding patterns at normal (pH 7.4) versus acidic (pH 6.0) conditions

• Application in Cancer Research: Similar to studies with other pH-dependent conjugates, DAPP1-FITC antibodies could potentially exploit the inherent acidity of solid tumors for selective binding. Research has shown pH-dependent increases in fluorescence levels when using fluorescent antibodies in acidic tumor environments .

• Methodology for pH-Selective Studies:

  • Treat cells with antibodies at both neutral (pH 7.4) and acidic (pH 6.0) conditions

  • Measure fluorescence by flow cytometry or fluorescence microscopy

  • Analyze pH-dependent differences in binding patterns and intensity

What are the optimal parameters for using DAPP1-FITC antibodies in multi-color flow cytometry?

When incorporating DAPP1-FITC antibodies into multi-color flow cytometry panels:

• Spectral Considerations:

  • FITC excitation maximum: 494 nm

  • FITC emission maximum: 519 nm

  • Compatible lasers: 488 nm (blue)

  • Avoid fluorophores with significant spectral overlap (e.g., GFP, Alexa Fluor 488)

• Panel Design Recommendations:

• Optimization Steps:

  • Perform single-color controls for accurate compensation

  • Titrate DAPP1-FITC antibody to determine optimal concentration

  • Include FMO (Fluorescence Minus One) controls to set accurate gates

• Technical Considerations:

  • FITC can photobleach during sorting; consider using reduced laser power

  • FITC fluorescence is pH-sensitive; maintain consistent buffer conditions

  • If studying cells with high autofluorescence, consider alternative fluorophores

How do phosphorylation states of DAPP1 affect antibody binding and detection?

DAPP1 function is regulated through phosphorylation, particularly at Tyr139, which affects its methodological detection using antibodies:

• Phosphorylation-Specific Detection:

  • Standard DAPP1-FITC antibodies typically recognize total DAPP1 regardless of phosphorylation state

  • Phospho-specific antibodies (e.g., anti-DAPP1 phospho-Tyr139) can be used to detect activated DAPP1

  • For comprehensive analysis, use both total and phospho-specific antibodies

• Experimental Considerations:

  • Insulin treatment (0.01U/ml for 2 minutes) induces DAPP1 phosphorylation as demonstrated in validation studies

  • Phosphatase inhibitors must be included in all buffers during sample preparation

  • Denaturing conditions in Western blotting may affect epitope accessibility differently for phosphorylated versus non-phosphorylated forms

• Dual Detection Strategy:

  • Use phospho-DAPP1 antibody conjugated to one fluorophore

  • Use total DAPP1-FITC antibody

  • Calculate the ratio of phosphorylated to total DAPP1 as a measure of activation

• Methodological Validation:

  • Treatment with phosphatase confirms specificity of phospho-antibody

  • Blocking peptides distinguishing between phosphorylated and non-phosphorylated epitopes

What are the advantages and limitations of using FITC versus other fluorophores for DAPP1 antibody conjugation?

When selecting FITC for DAPP1 antibody conjugation, consider these comparative advantages and limitations:

Methodological recommendation: If performing multi-parameter analysis or working in acidic environments, newer generation fluorophores may be preferable. For standard applications with neutral pH, FITC remains a cost-effective and well-characterized option with established protocols .

What methodological approaches can validate DAPP1-FITC antibody specificity in different experimental systems?

Comprehensive validation of DAPP1-FITC antibody specificity requires multiple complementary approaches:

• Western Blot Validation:

  • Verify single band at expected molecular weight (~32 kDa)

  • Perform peptide competition assay with immunizing peptide

  • Compare multiple antibodies targeting different DAPP1 epitopes

• Genetic Validation:

  • Test antibody in DAPP1 knockout/knockdown systems

  • Perform rescue experiments with DAPP1 overexpression

  • Compare antibody performance across species based on epitope conservation

• Cross-Reactivity Assessment:

  • Test in cells with varying DAPP1 expression levels

  • Evaluate potential cross-reactivity with structurally similar proteins

  • Confirm specificity across different sample types (cell lines, primary cells, tissues)

• Application-Specific Validation:

  • For flow cytometry: Compare surface vs. intracellular staining patterns

  • For IHC/ICC: Compare staining patterns with in situ hybridization results

  • For IP experiments: Confirm pull-down of known interaction partners

• Documentation Requirements:

  • Record lot-specific validation data

  • Maintain positive and negative control data

  • Document all optimization parameters (dilutions, incubation times, buffers)

How can DAPP1-FITC antibodies be used to study the role of DAPP1 in B-cell receptor signaling?

DAPP1-FITC antibodies provide valuable tools for investigating the dynamic role of DAPP1 in B-cell receptor (BCR) signaling pathways:

• Subcellular Localization Studies:

  • Track DAPP1 translocation following BCR stimulation using live-cell imaging

  • Co-localization experiments with other signaling components (e.g., PI3K, PIP3)

  • Compare wild-type versus mutant DAPP1 localization patterns

• Signaling Kinetics Analysis:

  • Time-course experiments measuring DAPP1 phosphorylation and localization

  • Correlation with downstream signaling events

  • Effect of pharmacological inhibitors on DAPP1 dynamics

• Methodological Protocol for BCR Stimulation Studies:

  • Isolate B cells and equilibrate in serum-free media

  • Stimulate BCR with anti-IgM (10 μg/ml) for varying time points (0-30 min)

  • Fix cells and permeabilize for intracellular staining with DAPP1-FITC antibody

  • Analyze by flow cytometry or confocal microscopy

• Functional Studies Integration:

  • Correlate DAPP1 localization/phosphorylation with calcium flux measurements

  • Integrate with phospho-specific antibodies against other signaling components

  • Connect to functional outcomes (proliferation, apoptosis, differentiation)

This approach provides comprehensive understanding of how DAPP1 regulates B-cell antigen receptor signaling downstream of PI3K activation .

How can researchers troubleshoot common issues with DAPP1-FITC antibody experiments?

When experiencing weak signals specifically in Western blotting, researchers should consider that some DAPP1 epitopes may be masked by protein folding or post-translational modifications. In such cases, different denaturing conditions or epitope retrieval methods may improve detection .