AGAP2 Antibody

What is AGAP2 and why are antibodies against it important for research?

AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2), also known as PIKE or Centaurin-gamma-1, is a multifunctional protein that plays crucial roles in cellular processes including endosomal trafficking, focal adhesion remodeling, and phagocytosis. The protein contains an N-terminal GTPase-like domain (GLD), a split PH domain, and a GTPase-activating protein (GAP) domain followed by four ankyrin repeats .

AGAP2 antibodies are essential research tools because they enable the detection, localization, and functional analysis of this protein across various experimental contexts. This is particularly important as AGAP2 has been implicated in several pathological conditions, including cancer, where its expression is often enhanced .

Which AGAP2 antibody applications are most widely validated?

AGAP2 antibodies have been validated across multiple applications with varying degrees of optimization:

Researchers should note that Western blot is generally the most robust application, while other techniques may require more extensive optimization depending on the specific antibody and experimental system .

What species reactivity should researchers expect with AGAP2 antibodies?

Most commercially available AGAP2 antibodies demonstrate confirmed reactivity against human, mouse, and rat AGAP2. This cross-reactivity is expected due to the high sequence homology between these species. For example, BLAST analysis of one AGAP2 antibody showed cross-reactivity with mouse and rat AGAP2 based on 93% and 95% protein homology, respectively, with the human immunizing sequence .

When evaluating an antibody's potential cross-reactivity with other species not explicitly tested, researchers should conduct homology analyses of the immunizing sequence against the target species' AGAP2 sequence to predict potential reactivity .

What are the optimal sample preparation protocols for AGAP2 detection in different applications?

For Western Blot analysis:

• Tissue samples: Homogenization in RIPA buffer with protease and phosphatase inhibitors is recommended

• Cell samples: Lysis in buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0) with protease inhibitors

• Load 20-50 μg of total protein per lane

• Use either 8% or 10% SDS-PAGE gels to properly resolve AGAP2 (observed MW: 124 kDa)

For Immunohistochemistry:

• Antigen retrieval is critical - recommended protocols include:

  • TE buffer pH 9.0 (primary method)

  • Alternative: citrate buffer pH 6.0

• Fixation with 4% paraformaldehyde is recommended

• Blocking with 5% normal goat serum reduces background staining

For Immunoprecipitation:

• Use 0.5-4.0 μg antibody for every 1.0-3.0 mg of total protein lysate

• Pre-clear lysates with Protein A/G beads before adding the antibody to reduce non-specific binding

How can researchers validate the specificity of an AGAP2 antibody?

A comprehensive validation approach should include multiple complementary methods:

• Positive and negative control tissues/cells:

  • Positive controls: Human brain tissue, rat brain tissue, HeLa cells

  • Negative controls: Tissues known to have minimal AGAP2 expression

• siRNA/shRNA knockdown verification:

  • Transfect cells with AGAP2-specific siRNA/shRNA (validated sequences: 5′-AGA CAC AUC UGG UGC UAA U-3′ and 5′-GUA AUG GCU UUC UAC UCU A-3′)

  • Compare antibody signals between knockdown and control samples via Western blot

  • Expect significant reduction in signal intensity in knockdown samples

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide

  • Apply to duplicate samples alongside untreated antibody

  • Specific signals should be abolished or significantly reduced

• Molecular weight verification:

  • AGAP2 should be detected at approximately 124-125 kDa

  • Note that some antibodies may detect AGAP2 at different apparent molecular weights (~90 kDa or ~42 kDa) depending on the isoform or potential proteolytic processing

What controls are essential when designing experiments with AGAP2 antibodies?

Robust experimental design requires these controls:

• Loading controls for Western blot:

  • Use housekeeping proteins (β-actin, GAPDH, tubulin) to normalize protein loading

  • For subcellular fractionation experiments, use compartment-specific markers (e.g., Lamin B for nuclear fraction)

• Isotype controls for IHC/IF:

  • Include normal rabbit IgG at the same concentration as the AGAP2 antibody

  • This controls for non-specific binding of the antibody class

• Tissue/cell specificity controls:

  • Include known AGAP2-positive tissues (brain) and AGAP2-negative tissues

  • Compare staining patterns across different tissues to confirm specificity

• Alternative antibody validation:

  • When possible, confirm results with a second AGAP2 antibody that recognizes a different epitope

  • This helps distinguish true signals from potential cross-reactivity

How can researchers effectively study AGAP2 phosphorylation in neutrophils?

AGAP2 undergoes phosphorylation in neutrophils upon stimulation with opsonized zymosan or monosodium urate crystals . To study this phosphorylation:

• Stimulation protocol:

  • Isolate fresh human neutrophils using density gradient centrifugation

  • Stimulate cells with either:

    • Opsonized zymosan (1 mg/ml)

    • Monosodium urate crystals (100 μg/ml)

  • Include time points from 0-30 minutes to capture phosphorylation kinetics

• Detection methods:

  • Immunoprecipitate AGAP2 from lysates using 2-4 μg of antibody per sample

  • Analyze by SDS-PAGE and Western blot with:
    a. Phospho-specific antibodies (if available)
    b. General phospho-serine/threonine/tyrosine antibodies
    c. Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

• Functional correlation:

  • Correlate phosphorylation status with phagocytosis efficiency using:

    • Fluorescent particle uptake assays

    • Live cell imaging of phagocytic cup formation

  • Compare results between neutrophils and neutrophil-like differentiated PLB-985 cells

• Phosphorylation site identification:

  • For advanced characterization, use mass spectrometry after immunoprecipitation

  • Focus on serine/threonine residues in the region between the PH domain and GAP domain

What approaches are recommended for studying the interaction between AGAP2 and focal adhesion kinase (FAK)?

The interaction between AGAP2 and FAK can be studied using these approaches:

• Co-immunoprecipitation optimization:

  • Use mild lysis conditions (1% NP-40, 150 mM NaCl, 50 mM Tris pH 7.4)

  • For bidirectional confirmation:
    a. Immunoprecipitate with anti-AGAP2 and blot for FAK
    b. Immunoprecipitate with anti-FAK and blot for AGAP2

  • The PH2 domain of AGAP2 is the primary binding site for FAK

• Domain mapping:

  • Generate GST fusion proteins containing different AGAP2 domains:

    • Full-length AGAP2

    • PH2 domain alone

    • AGAP2 without PH2 domain

  • Use GST pulldown assays to identify the minimal required interaction region

  • Compare binding efficiency between AGAP1 and AGAP2 (AGAP2 demonstrates stronger interaction)

• Functional analysis:

  • Transfect cells with:
    a. Wild-type AGAP2
    b. PH2 domain-deleted AGAP2

  • Assess effects on:

    • Focal adhesion formation (by paxillin/vinculin staining)

    • Cell migration (using Transwell assays)

    • FAK phosphorylation status (Y397 phosphorylation)

• Live-cell imaging:

  • Generate fluorescently tagged AGAP2 and FAK constructs

  • Perform FRET or BiFC analysis to visualize interaction dynamics in living cells

  • Correlate with focal adhesion assembly/disassembly events

How should researchers approach studying AGAP2's role in phagocytosis?

Based on recent findings about AGAP2's involvement in Fcγ receptor-mediated phagocytosis , researchers should consider:

• Cell model selection:

  • Primary human neutrophils (for physiological relevance)

  • PLB-985 cells differentiated toward neutrophil-like phenotype (for genetic manipulation)

  • CHO-IIA cells (stably expressing the FcγRIIA receptor) for reconstitution experiments

• Domain-specific function analysis:

  • Express domain-specific AGAP2 mutants:

    • GLD domain deletion (N-terminal GTP-binding protein-like domain)

    • GAP domain deletion

    • [R618K]AGAP2 (GAP-deficient but structurally intact)

  • Compare phagocytic efficiency across these constructs to distinguish between:
    a. GTPase activity requirements (not essential)
    b. GAP domain structural requirements (essential)
    c. GAP catalytic activity (not essential)

• Phagocytosis quantification methods:

  • Flow cytometry using fluorescent particles

  • Confocal microscopy with 3D reconstruction to distinguish attached vs. internalized particles

  • Live cell imaging to capture AGAP2 recruitment dynamics to phagocytic cups

• AGAP2 silencing approach:

  • Use validated siRNA sequences: 5′-AGA CAC AUC UGG UGC UAA U-3′ and 5′-GUA AUG GCU UUC UAC UCU A-3′

  • For stable knockdown, utilize lentiviral shRNA expression vectors

  • Include appropriate controls (non-targeting siRNA/shRNA)

Why might researchers observe different apparent molecular weights for AGAP2 in Western blot analyses?

Researchers frequently encounter variability in AGAP2's apparent molecular weight, which can be explained by several factors:

• Multiple isoforms:

  • AGAP2 exists in multiple isoforms (including PIKE-A, PIKE-L)

  • The calculated molecular weight is approximately 125 kDa

  • Observed weights range from 42 kDa to 124 kDa depending on the antibody and sample

• Post-translational modifications:

  • Phosphorylation can alter migration patterns

  • AGAP2 undergoes phosphorylation in response to stimuli such as opsonized zymosan

  • Compare untreated and stimulated samples to identify mobility shifts

• Proteolytic processing:

  • AGAP2 may undergo proteolytic processing in certain tissues/conditions

  • The 42 kDa band observed with some antibodies may represent a cleaved form

  • Use N- and C-terminal specific antibodies to distinguish full-length vs. processed forms

• Technical considerations:

  • Gel percentage affects resolution of high molecular weight proteins

  • Use 8% gels for optimal resolution of full-length AGAP2

  • Incomplete sample denaturation can cause aberrant migration

  • Include a positive control sample with confirmed AGAP2 expression (human brain tissue)

What approaches can resolve inconsistent or contradictory AGAP2 antibody results?

When facing inconsistent results across experiments, consider these methodological approaches:

• Antibody epitope mapping:

  • Determine the exact epitope recognized by each antibody

  • Different antibodies may recognize distinct domains or isoforms

  • Anti-PIKE antibodies may detect both PIKE-L and PIKE-A isoforms

• Sample preparation optimization:

  • Test multiple lysis buffers (RIPA vs. NP-40 vs. Triton X-100)

  • Include protease and phosphatase inhibitors

  • Standardize protein denaturation conditions (temperature, time, reducing agents)

• Cross-validation strategies:

  • Use multiple antibodies recognizing different AGAP2 epitopes

  • Employ orthogonal techniques (mass spectrometry)

  • Combine antibody detection with genetic approaches (overexpression, knockdown)

• Quantification considerations:

  • Establish a standardized densitometry approach

  • Use recombinant AGAP2 to create a standard curve

  • Report relative changes rather than absolute values when comparing across antibodies

How can researchers distinguish between specific and non-specific signals when using AGAP2 antibodies in immunohistochemistry?

Distinguishing specific from non-specific signals requires systematic approach:

• Antigen retrieval optimization:

  • Compare multiple antigen retrieval methods:

    • TE buffer pH 9.0 (primary recommended method)

    • Citrate buffer pH 6.0 (alternative method)

  • Optimize retrieval time and temperature for each tissue type

• Dilution series testing:

  • Perform a dilution series (1:50 to 1:500) for each new tissue type

  • Identify the dilution that maximizes specific signal while minimizing background

  • Document optimal dilutions for different tissues in standardized protocols

• Validation with knockout/knockdown tissues:

  • If available, use AGAP2 knockout or knockdown tissues as negative controls

  • Compare staining patterns to confirm specificity

  • For human samples where genetic models aren't available, use peptide competition assays

• Multi-technique confirmation:

  • Confirm IHC findings with IF on the same tissues

  • Validate protein expression with Western blot from the same samples

  • Consider RNAscope or in situ hybridization to confirm mRNA expression patterns

  • Compare antibody staining patterns with publicly available transcriptomic datasets

What methodologies are recommended for investigating AGAP2's role in cancer progression?

Recent research suggests AGAP2 overexpression in various cancers. To investigate this:

• Expression analysis across cancer types:

  • Analyze AGAP2 protein levels in tissue microarrays spanning multiple cancer types

  • Use the optimal IHC protocol (1:50-1:500 dilution with TE buffer pH 9.0 for antigen retrieval)

  • Correlate expression with clinical outcomes and pathological features

• Functional studies in cancer cell lines:

  • Generate stable AGAP2 knockdown cancer cell lines using validated shRNA sequences

  • Examine effects on:

    • Cell proliferation (using real-time cell analysis systems)

    • Migration and invasion (Transwell and Matrigel invasion assays)

    • Anchorage-independent growth (soft agar colony formation)

    • Tumor formation in xenograft models

• Signaling pathway analysis:

  • Focus on PI3K/Akt pathway activation status

  • Investigate interaction with FAK and subsequent downstream signaling

  • Use phospho-specific antibodies to monitor pathway activation

  • Perform rescue experiments with constitutively active pathway components

• AGAP2-AS1 antisense RNA studies:

  • Investigate the relationship between AGAP2 protein and its antisense RNA (AGAP2-AS1)

  • AGAP2-AS1 is upregulated in some cancers and may serve as a prognostic biomarker

  • Use RNA pulldown and RNA immunoprecipitation to identify protein interactions

How can researchers effectively study AGAP2 in exosomes and its potential as a biomarker?

Emerging research suggests AGAP2-AS1 presence in exosomes, which requires specialized techniques:

• Exosome isolation protocols:

  • Use differential ultracentrifugation (gold standard)

  • Alternative: commercial exosome isolation kits for higher throughput

  • Confirm exosome purification by:

    • Transmission electron microscopy

    • Nanoparticle tracking analysis

    • Western blot for exosomal markers (CD9, CD63, CD81)

• AGAP2/AGAP2-AS1 detection in exosomes:

  • For protein: Western blot using optimized AGAP2 antibodies (1:500-1:2000 dilution)

  • For RNA: qRT-PCR with specific primers for AGAP2-AS1

  • Compare exosomal content between:

    • Normal vs. cancer patient samples

    • Treatment-responsive vs. treatment-resistant patients

• Functional studies of exosomal AGAP2:

  • Isolate exosomes from cells with manipulated AGAP2 expression

  • Apply these exosomes to recipient cells

  • Monitor effects on recipient cell behavior:

    • Proliferation

    • Migration

    • Drug resistance

  • Track exosome uptake using fluorescent labeling techniques

• Clinical correlation studies:

  • Develop standardized protocols for exosome isolation from patient samples

  • Establish normalization methods for quantifying exosomal AGAP2/AGAP2-AS1

  • Correlate levels with disease progression, treatment response, and survival outcomes

What criteria should researchers use when selecting an AGAP2 antibody for specific applications?

When selecting an AGAP2 antibody, consider these application-specific criteria:

• For Western blot applications:

  • Prioritize antibodies with published Western blot validation

  • Confirm the detected molecular weight (should be ~124 kDa)

  • Review images in validation galleries for background levels

  • Recommended dilution ranges: 1:500-1:2000

• For immunohistochemistry:

  • Select antibodies specifically validated for IHC-P

  • Verify compatible antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Check validated dilution ranges (typically 1:50-1:500)

  • Review staining patterns in normal vs. disease tissues

• For immunofluorescence:

  • Confirm subcellular localization patterns match known AGAP2 distribution

  • Check if antibody has been validated in the specific cell type of interest

  • Typical dilution ranges: 1:20-1:200

• For immunoprecipitation:

  • Select antibodies specifically tested for IP applications

  • Verify amount needed (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Check if the antibody is supplied in a buffer compatible with IP

How can researchers design experiments to resolve contradictory findings in AGAP2 function?

To address contradictions in published AGAP2 functions:

• Systematic domain-function analysis:

  • Generate a complete set of domain-specific mutants:

    • GLD domain mutants/deletions

    • PH domain mutants/deletions

    • GAP domain mutants (catalytically inactive [R618K])

    • Ankyrin repeat mutants/deletions

  • Test each mutant across multiple functional assays (phagocytosis, focal adhesion dynamics, signaling)

• Cell type and context consideration:

  • Compare AGAP2 function across multiple cell types:

    • Phagocytic cells (neutrophils, macrophages)

    • Cancer cells (glioma, breast cancer)

    • Normal epithelial cells

  • Document cell type-specific differences in:

    • Expression levels (by Western blot)

    • Subcellular localization (by IF)

    • Protein interactions (by co-IP)

• Integrated multi-omics approach:

  • Combine:

    • Proteomics (interaction partners)

    • Phosphoproteomics (activation status)

    • Transcriptomics (downstream effects)

  • Analyze data using pathway enrichment tools to identify context-dependent functions

• Standardized reporting of experimental conditions:

  • Document in detail:

    • Cell culture conditions (media, serum, passage number)

    • Stimulation protocols (concentrations, timing)

    • Antibody sources, catalog numbers, and lot numbers

    • Image acquisition and analysis parameters