Quantitative analysis of annexin V–membrane

interaction by flow cytometry

Presented by Muhammad Younis

Background• Annexin A5 (or annexin V) is a cellular

protein in the annexin group.• AnxV can be used as an apoptosis marker.• In the presence of calcium, annexin V (anxV)

specifically binds to PS with high affinity, which can be used to detect PS externalization occurring on the membrane of apoptotic cells.

• To Distinguish early apoptotic cells from viable cells as well as late apoptotic and necrotic cells through flow cytometry.

Construction of pET28a(+)‑anxV‑EGFP‑his6 expression plasmid

Expression, purification, and characterizationof anxV‑EGFP

Preliminary characterization of anxV-EGFP protein.

Dotplot of R1 region of apoptotic cells

Calcium dependence of anxV-EGFP binding to apoptotic cell membrane

P: anxV-EGFP concentration , PBI: Protein binding capacity index,

Comparison between experimental and theoretical curves

Verification and further analyses of the mathematical expression.

Evaluation of the expression with anxV-FITC

Comparison between experimental curve and theoretical curve estimated under various concentrations of calcium and protein.

Summary• Constructed a green fluorescent

phosphatidylserine (PS)-binding probe, • With this probe,investigated anxV–membrane

interaction under different calcium and anxV-EGFP concentrations through flow cytometry (FCM).

• Comparison between the performance of anxV-EGFP and anxV-FITC provided further evidence in support of anxV-EGFP being an eligible and convenient probe for labeling externalized PS during apoptosis process.