Quantitative analysis of annexin V-membrane interaction by flow cytometry.
We constructed a green fluorescent phosphatidylserine (PS)-binding probe, which was generated by fusing enhanced green fluorescent protein (EGFP) to the C terminus of human annexin V (anxV). With this probe, we investigated anxV-membrane interaction under different calcium and anxV-EGFP concentrations through flow cytometry (FCM). A mathematical description of the binding characteristics is proposed and validated to quantify the relationship concerning the relative concentration of membrane-bound anxV (B), calcium concentration ([C]), and protein concentration ([P]). Further analyses reveal that [Formula: see text] is linear with [Formula: see text] or [Formula: see text] when [P] and [C] are fixed, respectively, which indicates that the anxV-membrane binding reaction may involve sequential multiple steps. Our study provides a reference for application of anxV in apoptosis detection. The mathematical expression facilitates exploration of the possible interactions between calcium, anxV, and membrane. The corresponding mathematical analysis strengthens the interpretation of the interaction data.
The State Key Laboratory of Pharmaceutical Biotechnology, Room B211, College of Life Sciences, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, 163 Xianlin Road, 210046, Nanjing, People's Republic of China.
Jie
J
Wang
Liangqiang
L
He
Dianhua
D
Chen
Yazhou
Y
Pi
Wenping
W
Zhou
Xingkui
X
Xiong
Yongzhe
Y
Ren
Yueyang
Y
Lai
Zichun
Z
Hua
D017304:Annexin A5 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008962:Models, Theoretical; D011485:Protein Binding
D017304:Annexin A5; D002118:Calcium