Date post: | 11-Feb-2017 |
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Science |
Upload: | muhammad-younis |
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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.