DAPP1 Antibody

What is DAPP1 and why is it important in immune cell research?

DAPP1 (Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides 1, also known as BAM32) is a 31-32 kDa adaptor protein that plays a critical role in B cell receptor (BCR) signaling. It shows restricted expression, being primarily found in:

• Mast cells

• Dendritic cells

• Germinal center B cells

DAPP1's importance stems from its dual-domain structure containing both SH2 and PH domains, enabling it to coordinate membrane-localized interactions among proteins from distinct signal transduction pathways. When B cell receptors engage with antigens, PI3-kinase activates and generates membrane-embedded PI(3,4)P2, which serves as a ligand for cytosolic DAPP1. This interaction results in DAPP1's immobilization at the cell membrane, where it undergoes phosphorylation on Tyr139 .

Functionally, DAPP1 is involved in:

• BCR internalization

• Antibody isotype switching

• Antigen processing and presentation

• B cell survival

Research using DAPP1 knockout mice has demonstrated that while B and T cell development remains normal, B cell proliferation is reduced by approximately 50% after BCR crosslinking compared to wild-type mice. Additionally, these mice show defective T-independent type II immune responses and impaired antibody production against bacterial polysaccharides .

What applications are DAPP1 antibodies commonly used for in research?

Based on the available antibody products, DAPP1 antibodies have been validated for multiple experimental applications:

For optimal results, it's recommended that each laboratory determine the optimal dilutions for their specific application and experimental conditions .

What species reactivity is available for DAPP1 antibodies, and how can cross-reactivity be confirmed?

Commercial DAPP1 antibodies are available with reactivity to various species:

When confirming cross-reactivity between species, consider that human DAPP1 shares approximately 91% amino acid sequence identity with mouse DAPP1 over amino acids 1-163 . This high homology suggests antibodies raised against this region will likely recognize both human and mouse proteins, as demonstrated by Western blot results showing detection of DAPP1 in both human cell lines (A431, Daudi, Ramos) and mouse cell lines (BaF3) .

To experimentally confirm cross-reactivity:

• Run Western blots with positive control lysates from both species

• Include negative controls (knockout samples or tissues not expressing DAPP1)

• Verify that the molecular weight is consistent between species (approximately 32 kDa)

• For phospho-specific antibodies, treat samples with phosphatase to confirm specificity

How should DAPP1 antibodies be stored and handled to maintain optimal activity?

Based on manufacturer recommendations across multiple products, the following storage and handling guidelines should be followed:

Short-term storage:

• Reconstituted antibodies: 2-8°C for 1-2 weeks

Long-term storage:

• Lyophilized/supplied form: -20°C to -70°C for up to 12 months from date of receipt

• Reconstituted antibodies: -20°C to -70°C for up to 3-6 months in appropriate storage buffer

Critical handling notes:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• When reconstituting lyophilized antibodies:

  • Centrifuge vial before opening

  • Gently pipet and wash down the sides of the vial to ensure full recovery

  • For some products, adding an equal volume of glycerol is recommended for long-term storage

• Aliquot reconstituted antibodies to minimize freeze-thaw cycles

• Some products contain sodium azide as a preservative which should be handled by trained staff only

How does phosphorylation of DAPP1 at Tyr139 affect its function, and what are the best methods to study this modification?

Phosphorylation of DAPP1 at Tyr139 is a key regulatory event that occurs following B cell receptor (BCR) activation. This phosphorylation:

• Is PI3K-dependent and requires an intact PH domain in DAPP1

• Is likely performed by Src-family kinases following membrane recruitment of DAPP1 by phosphoinositides

• Directly regulates HPK1 (hematopoietic progenitor kinase 1) activity

• Indirectly regulates HPK1 downstream targets ERK and JNK

• Plays a critical role in regulating actin-dependent internalization processes

Research has shown that blocking phosphorylation of DAPP1 at Tyr139 inhibits BCR internalization and reduces cellular F-actin levels, indicating its importance in cytoskeletal reorganization during immune responses .

Methodological approaches to study Tyr139 phosphorylation:

• Phospho-specific antibodies:

  • Cell Signaling's Phospho-DAPP1/BAM32 (Tyr139) (D7G4G) Rabbit mAb (#13703) specifically detects endogenous DAPP1 phosphorylated at Tyr139

  • Recommended dilutions: 1:1000 for Western blotting, 1:200 for immunoprecipitation

• Experimental design considerations:

  • Include both unstimulated and BCR-stimulated B cells

  • Use PI3K inhibitors (e.g., wortmannin) to demonstrate PI3K-dependency

  • Include phosphatase treatment as a negative control

  • Consider using DAPP1 mutants (Y139F) for functional studies

• Complementary techniques:

  • Combine phospho-specific Western blotting with immunofluorescence to assess subcellular localization

  • Use co-immunoprecipitation to identify interaction partners dependent on Tyr139 phosphorylation

  • Employ live-cell imaging with fluorescently tagged DAPP1 to monitor translocation dynamics

What controls should be included when using DAPP1 antibodies in signaling pathway studies?

When investigating DAPP1's role in signaling pathways, particularly in B cell receptor (BCR) signaling, comprehensive controls are essential for rigorous experimental design:

Positive Controls:

• Cell lines with known DAPP1 expression:

  • Human: A431 (epithelial carcinoma), Daudi (Burkitt's lymphoma), Ramos (Burkitt's lymphoma), IM-9

  • Mouse: BaF3 (pro-B cell line)

• Tissue samples with high DAPP1 expression:

  • Human: Lymphoid tissues, especially germinal centers

  • Mouse: Splenic B cells

• Stimulation conditions:

  • BCR crosslinking with anti-IgM/anti-IgG antibodies

  • PI3K activation with appropriate growth factors

Negative Controls:

• DAPP1 knockout or knockdown samples:

  • DAPP1-/- mice B cells

  • siRNA/shRNA-treated cells

• Cell lines with low/no DAPP1 expression:

  • T cells show lower expression compared to B cells

  • Non-hematopoietic cells typically have minimal expression

• Blocking peptides:

  • Pre-incubation of antibody with immunizing peptide/protein

Treatment Controls:

• For phosphorylation studies:

  • λ-phosphatase treatment to remove phosphorylation

  • PI3K inhibitors (wortmannin, LY294002) to block DAPP1 membrane recruitment

• Time course experiments:

  • Include multiple time points after stimulation (0, 1, 5, 15, 30 min)

  • Monitor both phosphorylation and subcellular localization changes

• Antibody specificity validation:

  • Use multiple antibodies recognizing different epitopes

  • Compare monoclonal (MAB7024) vs polyclonal (AF7024) antibody results for confirmation

What are the key differences between polyclonal and monoclonal DAPP1 antibodies for specific applications?

The choice between polyclonal and monoclonal DAPP1 antibodies significantly impacts experimental outcomes. Below is a comparative analysis based on available DAPP1 antibody products:

Application-specific recommendations:

• For Western blotting:

  • Monoclonal antibodies like MAB7024 or ab131212 provide cleaner results with specific bands at 32 kDa

  • Recommended dilutions: 1:500-1:1000

• For Immunohistochemistry:

  • Polyclonal antibody #109864 has been validated for human liver cancer tissue

  • Recommended dilutions: 1:20-1:200

• For phospho-DAPP1 detection:

  • Monoclonal antibody #13703 specifically recognizes phosphorylation at Tyr139

  • Essential for studying BCR signaling dynamics

• For multi-species studies:

  • Antibodies AF7024 and MAB7024 detect both human and mouse DAPP1 in Western blots

What methods are most effective for validating the specificity of DAPP1 antibodies?

Validating antibody specificity is crucial for obtaining reliable results in DAPP1 research. A comprehensive validation approach should include:

1. Genetic Models:

• Positive approach: Compare wild-type vs. DAPP1 knockout samples (e.g., Bam32-/- mice)

• Methodology: Western blot analysis of B cells from wild-type and DAPP1-deficient mice should show absence of the 32 kDa band in knockout samples

2. Expression Systems:

• Recombinant protein validation:

  • Use purified DAPP1 proteins as positive controls:

    • E. coli-derived recombinant human DAPP1 (Gly2-Ser163)

    • Full-length DAPP1 (1-280 aa) with tags (GST, Strep)

  • Compare endogenous vs. overexpressed protein

3. Orthogonal Methods:

• Multiple detection techniques:

  • Compare protein detection by mass spectrometry with antibody-based methods

  • Correlate protein expression with mRNA levels using RT-PCR

4. Multiple Antibodies:

• Epitope comparison:

  • Use antibodies recognizing different regions of DAPP1:

    • N-terminal region antibodies (including SH2 domain)

    • C-terminal region antibodies (including PH domain)

  • Verify consistent molecular weight detection (32 kDa)

5. Blocking Peptides:

• Competition assays:

  • Pre-incubate antibody with immunizing peptide/protein

  • Signal should be abolished or significantly reduced

6. Phospho-specific Validation:

• For phospho-DAPP1 (Tyr139) antibodies:

  • Treat samples with λ-phosphatase

  • Compare stimulated vs. unstimulated cells

  • Use Y139F DAPP1 mutant as negative control

7. Cross-reactivity Assessment:

• Species comparison:

  • Human DAPP1 shares ~91% amino acid sequence identity with mouse DAPP1 over aa 1-163

  • Confirm expected bands in multiple species using Western blot

How can DAPP1 antibodies be effectively used to study alternative splice variants of DAPP1?

DAPP1 has several potential alternative splice variants that researchers should consider when designing experiments:

Known DAPP1 splice variants:

• Five amino acid substitution for aa 259-280

• 22 amino acid substitution for aa 259-280

• 14 amino acid substitution for aa 1-229

• Deletions of aa 35-75 and aa 180-200 coupled to a three amino acid substitution for aa 249-280

• An additional isoform with a 22 amino acid substitution at the C-terminus

Methodological approaches for studying splice variants:

• Antibody selection strategy:

  • Choose antibodies targeting conserved vs. variant-specific regions:

    • Antibodies raised against aa 1-163 (e.g., AF7024, MAB7024) will detect most variants

    • C-terminal targeting antibodies may be variant-specific

• Western blot optimization:

  • Use gradient gels (4-20%) to better resolve variants with small size differences

  • Extended run times to separate closely migrating bands

  • Consider 2D gel electrophoresis for complex samples

• Detection techniques:

  • Combine protein and mRNA analysis:

    • Western blot for protein detection

    • RT-PCR with variant-specific primers

    • Correlate protein bands with transcript variants

• Expression system approach:

  • Generate expression constructs for each variant

  • Create positive controls using tagged variants

  • Compare migration patterns with endogenous proteins

• Sample considerations:

  • Analyze DAPP1 expression across various tissues and cell types

  • Compare different B cell developmental stages

  • Assess expression changes under different stimulation conditions

Example experimental workflow:

• Design primers to amplify full-length and variant DAPP1 transcripts

• Create expression constructs for each variant

• Perform Western blots using antibodies targeting different regions

• Create a migration pattern reference table for each variant

• Compare endogenous bands from different tissues/cells to reference patterns

What are the best protocols for using DAPP1 antibodies in studying B cell receptor (BCR) signaling dynamics?

Studying BCR signaling dynamics with DAPP1 antibodies requires careful experimental design that captures the temporal and spatial regulation of this adaptor protein. Based on the functional characteristics of DAPP1 in BCR signaling, here is a comprehensive protocol approach:

Sample Preparation and Stimulation:

• B cell isolation and culture:

  • Isolate primary B cells from human or mouse sources using magnetic separation

  • Alternatively, use B cell lines with confirmed DAPP1 expression:

    • Human: Daudi, Ramos (Burkitt's lymphoma cell lines)

    • Mouse: BaF3 (pro-B cell line)

• BCR stimulation conditions:

  • Optimal timing: Create a time course (0, 1, 2, 5, 10, 15, 30 min)

  • Stimulation methods:

    • Anti-IgM F(ab')₂ fragments (10 μg/ml)

    • Anti-IgG for class-switched B cells

    • For comparison, include CD19 stimulation or PI3K activators

Immunoblotting Protocol for Phosphorylation Analysis:

• Lysis conditions:

  • Use Immunoblot Buffer Group 1 as referenced in product data sheets

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride)

  • Perform lysis under reducing conditions

• Antibody selection and dilutions:

  • Total DAPP1: Anti-DAPP1 antibody at 1:1000 dilution

  • Phospho-DAPP1: Phospho-DAPP1/BAM32 (Tyr139) antibody at 1:1000 dilution

  • Secondary antibodies:

    • For sheep primary: HRP-conjugated Anti-Sheep IgG (HAF016)

    • For mouse primary: HRP-conjugated Anti-Mouse IgG (HAF007)

    • For rabbit primary: HRP-conjugated Anti-Rabbit IgG

• Technical considerations:

  • Load 10-35 μg of protein per lane

  • Include phosphorylation state controls (phosphatase-treated samples)

  • DAPP1 appears at approximately 32 kDa, with phosphorylated forms showing slightly higher mobility (34-36 kDa)

Subcellular Localization Studies:

• Immunofluorescence protocol:

  • Fix cells with 4% paraformaldehyde

  • Use DAPP1 antibody at 1:50 dilution

  • Co-stain with membrane markers to assess translocation

  • Counterstain nucleus with DAPI

• Fractionation approach:

  • Separate cytosolic and membrane fractions before and after BCR stimulation

  • Perform Western blots for DAPP1 on each fraction

  • Analyze recruitment kinetics from cytosol to membrane

Analysis of DAPP1-Dependent Signaling:

Compare phosphorylation of known downstream targets in the presence/absence of DAPP1:

• HPK1 activation

• ERK and JNK pathway components

• Actin cytoskeleton regulators involved in BCR internalization

This comprehensive approach provides a methodological framework for using DAPP1 antibodies to dissect the complex dynamics of B cell receptor signaling.