CD2AP Antibody

What is CD2AP and why is it important in research?

CD2AP is an adaptor protein that plays critical roles in regulating signal transduction and cytoskeletal molecules. It's ubiquitously expressed with higher levels in immune cells, epithelial cells, and neurons. Research significance stems from its involvement in multiple pathways including endocytosis, intercellular junction formation, and association with neurodegenerative diseases, particularly Alzheimer's disease (AD) . Recent evidence also suggests CD2AP promotes glioblastoma multiforme (GBM) progression via TRIM5-mediated NF-κB signaling .

What are the key applications for CD2AP antibodies in research?

CD2AP antibodies are primarily used in Western blotting (1:1000-1:20000 dilutions), immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg total protein), immunofluorescence (1:50-1:500), and co-immunoprecipitation studies . These applications enable researchers to investigate CD2AP protein expression, localization, and protein-protein interactions in various experimental models.

Which tissue types and cell lines show consistent CD2AP expression?

CD2AP expression has been reliably detected in:

How should CD2AP antibodies be validated for specific applications?

Proper validation should include:

• Positive and negative controls (CD2AP knockout or knockdown tissues)

• Tissue-specific expression patterns (e.g., glomerular basement membrane in kidney)

• Molecular weight verification (~70-80 kDa band in Western blot)

• Cross-reactivity testing across species

For example, the Proteintech CD2AP antibody (51046-1-AP) was validated using Lentiviral-mediated CD2AP-knockdown in hTERT-RPE1 and NIH3T3 cells, confirming specificity by the absence of signal in knockdown tissues .

What are the optimal conditions for immunofluorescence detection of CD2AP in neuronal tissues?

For neuronal tissues, particularly in studying cholinergic neurons or brain regions relevant to Alzheimer's disease:

• Use fresh, perfusion-fixed tissue (4% paraformaldehyde)

• Employ antigen retrieval methods if necessary

• Use 1:50-1:500 dilution of primary antibody with overnight incubation at 4°C

• Include colocalization markers for specific neuronal populations (e.g., ChAT for cholinergic neurons)

• Include Rab5 co-staining to investigate endocytosis mechanisms

What factors influence Western blot detection of CD2AP?

Several factors can impact CD2AP detection in Western blotting:

• Sample preparation: Reducing conditions are recommended (as noted in R&D Systems' protocol)

• Dilution range: Wide range (1:1000-1:20000) depending on antibody source and sample type

• Expected molecular weight: CD2AP typically appears at approximately 70-80 kDa

• Buffer selection: Immunoblot Buffer Group 1 has been validated for certain antibodies

• Secondary antibody selection: HRP-conjugated Anti-Sheep IgG or species-appropriate alternatives based on the primary antibody host

How does CD2AP contribute to Alzheimer's disease pathogenesis?

Current evidence suggests multiple mechanisms:

• CD2AP loss of function leads to enhanced Aβ metabolism and production

• CD2AP modulates Tau-induced neurotoxicity (homolog demonstrated in Drosophila models)

• CD2AP affects synaptic dysfunction and abnormal neurite structure

• CD2AP maintains blood-brain barrier integrity, and its compromise may accelerate disease

• Several single nucleotide polymorphisms (SNPs) in the CD2AP gene are associated with increased AD risk

• mRNA levels of CD2AP are decreased in peripheral lymphocytes of sporadic AD patients

What experimental approaches are recommended for studying CD2AP's role in the blood-brain barrier?

To investigate CD2AP's role in blood-brain barrier (BBB) integrity:

• Use CD2AP knockout/knockdown models with nephrin promoter-driven CD2AP transgene to prevent early mortality from renal disease

• Employ BBB permeability assays using labeled tracers

• Assess tight junction protein expression and localization

• Compare CD2AP expression in cerebrovascular endothelial cells versus neurons

• Consider age-dependent effects (2.5-4.5 months shown in previous studies)

What is known about CD2AP's interaction with endocytic pathways in neurons?

CD2AP has been shown to positively regulate Rab5-mediated mechanisms of endocytosis in primary sensory neurons. Immunofluorescence analysis reveals near-complete overlap of CD2AP and Rab5 expression in cholinergic neurons of the basal forebrain region, suggesting CD2AP may orchestrate endocytosis and retrograde signaling mechanisms relevant to neurodegenerative disorders . This relationship is particularly important when investigating how CD2AP variants might affect mechanistic components of Rab5 endocytosis.

How can researchers investigate CD2AP's role in glioblastoma progression?

Recent evidence suggests CD2AP promotes GBM progression through TRIM5-mediated NF-κB signaling. Recommended approaches include:

• Analyze CD2AP expression in GBM clinical samples and cell lines

• Perform CD2AP overexpression and knockdown experiments to assess effects on:

  • Cell proliferation and colony formation

  • Migration and invasion

  • Response to temozolomide treatment

  • Apoptosis rates

• Examine protein-protein interactions via co-immunoprecipitation (2397 proteins were identified as interacting with CD2AP)

• Focus on the CD2AP-TRIM5-NF-κB axis as a potential therapeutic target

• Assess patient prognosis correlation with CD2AP expression levels

What methodologies are recommended for studying CD2AP in cholinergic neurons?

For investigating CD2AP in cholinergic neurons:

• Use ChATBAC-eGFP transgenic mice where cholinergic neurons express GFP

• Perform perfusion and tissue harvesting of brain regions relevant to AD pathogenesis

• Conduct immunofluorescence analysis with appropriate antibodies:

  • Anti-CD2AP: Proteintech Cat# 51046-1-AP (validated in previous studies)

  • Anti-Rab5 for colocalization studies

  • Anti-ChAT to confirm cholinergic neuron identity

• Analyze expression patterns across different brain regions, particularly the diagonal band of Broca and basal forebrain

• Quantify signal intensity and colocalization metrics for comparative analysis

What approaches should be used when investigating contradictory findings between CD2AP mRNA and protein expression levels?

When faced with discrepancies between mRNA and protein data:

• Directly compare RNA-seq/qPCR data with protein quantification in the same samples

• Consider cell/tissue type-specific expression patterns (e.g., 5-fold higher expression in endothelial cells compared to neurons)

• Evaluate post-transcriptional and post-translational regulatory mechanisms

• Assess protein stability and turnover rates in different cellular compartments

• Verify antibody specificity to rule out detection artifacts

• Analyze temporal dynamics of expression, as mRNA and protein levels may not correlate at all time points

What are common issues in CD2AP antibody applications and their solutions?

How should researchers interpret differences in CD2AP localization across tissue types?

CD2AP localization varies significantly between tissues, requiring careful interpretation:

• In kidney: Primarily localized to podocyte foot processes and the glomerular basement membrane

• In brain: Mostly in cerebrovascular endothelial cells, with lower expression in neurons

• In immune cells: Associated with the T-cell receptor complex and immunological synapse

• In epithelial cells: Found at cell-cell junctions and associated with cytoskeletal structures

When unexpected localization patterns are observed, verify with multiple antibodies and confirm with knockout/knockdown controls to rule out non-specific binding .

What considerations are important when designing experiments to study CD2AP variants in Alzheimer's disease models?

When investigating CD2AP variants in AD models:

• Utilize humanized CD2AP variant models (such as those created by MODEL-AD)

• Consider both haploinsufficiency and genetic variant models

• Assess both cellular phenotypes (endocytosis, Aβ production, tau toxicity) and behavioral outcomes

• Include age-matched controls and temporal progression analysis

• Combine with other AD risk factors to assess synergistic effects

• Focus on cell type-specific effects (neurons vs. endothelial cells)

• Measure both CD2AP expression levels and functional outcomes in multiple brain regions relevant to AD