APBA2 Antibody

How do researchers validate APBA2 antibody specificity in Western blot assays?

Validation of APBA2 antibody specificity requires a multi-step approach. First, researchers should confirm the antibody’s reactivity using knockout (KO) controls or siRNA-mediated APBA2 knockdown lysates. For example, the absence of bands in APBA2-deficient tissues or cell lines confirms specificity . Second, peptide blocking assays are critical: pre-incubating the antibody with its immunogen peptide should abolish signal . Third, cross-reactivity profiling is essential. Thermo Fisher’s PA5-47830 antibody demonstrates <1% cross-reactivity with APBA1/APBA3 isoforms, validated via direct ELISA . Researchers should also compare observed molecular weights to theoretical predictions (e.g., 83 kDa predicted vs. 120–135 kDa observed due to post-translational modifications) .

Table 1: Validation Parameters for Select APBA2 Antibodies

What positive controls are recommended for APBA2 detection across experimental platforms?

Human brain tissue lysates (cortex or hippocampus) serve as gold-standard positive controls due to APBA2’s neuronal expression . For cell-based studies, overexpression systems (e.g., HEK293T transfected with APBA2 plasmids) are optimal . In Western blotting, R&D Systems’ AF6327 antibody detects APBA2 in human, mouse, and rat brain lysates, with stronger signals in cortical vs. hippocampal extracts . For immunohistochemistry, formalin-fixed paraffin-embedded (FFPE) brain sections show distinct neuronal staining patterns . Researchers should include recombinant APBA2 protein (e.g., residues 2–165 for AF6327 ) as a migration control to address anomalous electrophoretic mobility .

How do observed molecular weight discrepancies impact interpretation of APBA2 immunoblot data?

APBA2 migrates anomalously at 120–135 kDa in SDS-PAGE despite a predicted 83 kDa molecular weight . This discrepancy arises from two factors: (1) extensive post-translational modifications (e.g., phosphorylation at Ser238) , and (2) splice variants. Proteintech’s 19781-1-AP antibody detects isoforms with 44 aa substitutions (aa 170–214) and truncations (Δ406–417) . Researchers must correlate immunoblot data with mRNA splice variant expression (e.g., NM_005503 vs. NM_001368357) and use isoform-specific controls. For example, the N722S autism-associated mutation alters APBA2’s PDZ domain structure, affecting neurexin-1α binding without changing apparent MW .

What strategies address cross-reactivity challenges when studying APBA2 orthologs in comparative models?

Cross-species reactivity varies significantly. Boster Bio’s A06783 reacts with human, mouse, and rat APBA2 , while PA5-21053 is human-specific . Epitope mapping is critical: antibodies targeting conserved regions (e.g., PA5-47830 against aa 366–533 PTB domain ) show broader reactivity. Researchers should validate cross-species reactivity using:

• Ortholog sequence alignment: Mouse APBA2 shares 85% identity with human within residues 1–165 .

• Functional assays: Co-immunoprecipitation (co-IP) of APBA2 with APP in murine models confirms antibody utility .

• Blocking peptides: Species-specific blocking peptides eliminate off-target binding (e.g., PEP-1167 for PA5-21053 ).

Table 2: Cross-Reactivity Profiles of APBA2 Antibodies

How can APBA2 antibodies elucidate molecular mechanisms in Alzheimer's disease pathogenesis?

APBA2 stabilizes amyloid precursor protein (APP) and inhibits Aβ peptide production . To study this:

• Co-IP experiments: Use APBA2 antibodies (e.g., PA5-47830) to pull down APP complexes from brain lysates .

• Subcellular localization: Immunofluorescence with antibodies like ab137888 reveals APBA2-APP colocalization in endosomes .

• Functional knockdown: siRNA + APBA2 antibody staining quantifies Aβ accumulation in neuronal cultures .
  The PA5-47830 antibody detects APBA2 in Alzheimer’s patient brain sections, showing reduced expression in plaques vs. healthy tissue .

What advanced techniques combine APBA2 antibodies with functional studies of autism-linked mutations?

The autism-associated N722S mutation disrupts APBA2’s PDZ domain interactions . Methodologies include:

• Live-cell imaging: Transfect neurons with GFP-tagged APBA2 mutants and track synaptic vesicle trafficking using antibodies like AF6327 .

• Surface biotinylation: PA5-21053 quantifies membrane-bound neurexin-1α in HEK293T cells co-expressing wild-type/mutant APBA2 .

• Electrophysiology: Pair APBA2 immunostaining (e.g., A06783 ) with patch-clamp recordings to assess synaptic transmission deficits in knock-in models.

Key Finding: The N722S mutation reduces neurexin-1α surface expression by 40% compared to wild-type APBA2, implicating trafficking defects in autism pathogenesis .

Methodological Recommendations

• Multiplex validation: Combine KO controls, peptide blocking, and orthogonal techniques (e.g., mRNA correlation).

• Isoform-specific analysis: Use antibodies targeting variable regions (e.g., A06783 against aa 371–420 ) to distinguish splice variants.

• Dynamic range optimization: Titrate antibodies like PA5-47830 across 1:500–1:2000 dilutions to avoid saturation in high-expression tissues .