ECA1 Antibody

ECA1 Antibody Development and Validation

ECA1 antibodies have been generated against specific epitopes of the ECA1 protein:

• Polyclonal antibody (anti-ACA3 1374): Developed using a glutathione S-transferase fusion protein containing the C-terminal 27 residues of Arabidopsis ECA1. This antibody detected ECA1 in microsomal membranes via Western blotting .

• Carboxyl-tail-targeted antibody: Used to confirm the endoplasmic reticulum (ER) localization of ECA1 in Arabidopsis and yeast, demonstrating its role in calcium transport .

Calcium Pump Activity

• Yeast complementation: Expression of Arabidopsis ECA1 in yeast mutants (pmr1 pmc1 cnb1) restored growth under calcium-depleted conditions. Immunoblotting with ECA1 antibodies confirmed protein expression in transformed yeast .

• Calcium transport kinetics: ECA1 exhibited high-affinity Ca²⁺ pumping (Km = 30 nM) in yeast vesicles, as validated by ⁴⁵Ca²⁺ uptake assays .

Role in Stress Tolerance

• Cryptococcus neoformans: ECA1 antibodies helped identify ECA1’s role in fungal virulence and thermotolerance. Deletion mutants (eca1Δ) showed hypersensitivity to ER stress and calcineurin inhibitors, with reduced survival in macrophage infection models .

Key Research Applications

ECA1 antibodies have been critical for:

• Protein localization: Confirming ER membrane association in Arabidopsis and yeast .

• Mutant validation: Detecting ECA1 expression loss in C. neoformans eca1Δ strains via Northern and Western blots .

• Functional assays: Linking ECA1 to calcium signaling pathways parallel to calcineurin in fungal pathogens .

Technical Considerations

• Cross-reactivity: Antibodies raised against Arabidopsis ECA1 do not cross-react with human or fungal ECA1 homologs due to sequence divergence.

• Assay compatibility: Effective in Western blotting, immunostaining, and functional studies in yeast and plant models .

Implications and Future Directions

ECA1 antibodies have clarified conserved mechanisms of calcium signaling across species. Future work may explore:

• Therapeutic targeting of ECA1 in fungal infections .

• ECA1’s role in cancer progression via Wnt pathway modulation .