GASA3 Antibody

Basic Research Questions

How to validate GABRA3 antibody specificity in Western blot and immunohistochemistry?

Methodological Answer:

• Western Blot:

  • Use transfected COS-7 cells overexpressing GABRA3 as a positive control and non-transfected cells as a negative control. A band at ~50–60 kDa (expected molecular weight) should appear only in transfected lysates .

  • Preincubate the antibody with its immunizing peptide (e.g., #BLP-GA003) to confirm signal loss, ensuring epitope specificity .

• Immunohistochemistry:

  • Compare staining patterns in wild-type tissues vs. tissues from GABRA3-knockout models (e.g., cerebellar molecular layer in rats) .

  • Use secondary antibody-only controls to rule out nonspecific binding.

What experimental designs are optimal for detecting cell-surface GABRA3 in live cells?

Methodological Answer:

• Flow Cytometry:

  • Use intact, non-permeabilized cells (e.g., mouse BV-2 microglia or rat RIN-m insulinoma cells) to preserve extracellular epitopes.

  • Combine with AlexaFluor-conjugated secondary antibodies for quantitative analysis .

• Live-Cell Imaging:

  • Apply antibodies to living cells at 1:50–1:100 dilution, followed by fixation and counterstaining for subcellular markers (e.g., neurofilament 200 for neuronal processes) .

Advanced Research Questions

How to resolve discrepancies in GABRA3 localization across different cellular models?

Methodological Answer:

• Context-Specific Staining:

  • GABRA3 exhibits tissue-specific glycosylation (e.g., sialic acid modifications in erythroid cells ). Treat cells with neuraminidase to determine if epitope accessibility depends on post-translational modifications.

  • Compare antibody reactivity in primary cells (e.g., Purkinje neurons ) vs. immortalized lines (e.g., K562 erythroleukemic cells ).

• Cross-Validation:

  • Use orthogonal methods like RNAscope® (to confirm mRNA expression) or CRISPR-Cas9 knockout lines to validate protein localization .

How can computational tools like AlphaFold3 improve GABRA3 antibody-antigen interaction modeling?

Methodological Answer:

• Epitope Mapping:

  • Input GABRA3’s extracellular domain (residues 29–43 ) into AlphaFold3 to predict paratope-epitope docking. Validate predictions via mutagenesis (e.g., alanine scanning of CDR regions) .

• Structural Refinement:

  • Combine cryo-EM data of GABRA3-antibody complexes with AlphaFold3’s predictions to resolve conformational dynamics (median CDR H3 RMSD: 1.14–2.04 Å in nanobodies ).

Data Tables

Table 1: Key Validation Steps for GABRA3 Antibodies

Table 2: Species Reactivity and Technical Considerations

Contradiction Analysis

Why do some studies report GABRA3 in non-neuronal tissues (e.g., erythroid cells)?

• Hypothesis: Cross-reactivity with sialylated epitopes shared across lineages (e.g., leukosialin in erythroid cells ).

• Resolution: Perform glycan cleavage (neuraminidase) or use knockout models to confirm target specificity .

How to address low antibody sensitivity in endogenous expression systems?

• Solution: Optimize antigen retrieval (e.g., citrate buffer for IHC) or combine with signal amplification systems (e.g., tyramide-based kits) .