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Measuring site density of P-selectin on planar surface based on 125I labeling

Author(s): Yingchen Ling, Jinhe Zhang, Quhuan Li, Bing Huang, Ying Fang

Measuring the site density of P-selectin on planar substrate is the primary event in understanding the mechanismof P-selectin-induced cell adhesion onmolecular level through flowchamber experiment. In order to overcome two major disadvantages of current measurement means: low signal noise ratio of fluorescence labels and the limitation of traditional gamma counter or scintillation counter, herein, a novel method based on combination of radioactively 125I labels and Infinia Hawkeye 4 ECT is proposed to detect the low density of the P-selectin on planar surface. The results illustrate that site density of P-selectin is linear with their concentration, and glass plate shows better adsorption than polystyrene. For the adsorbed Pselectin on the plate at same concentration, the difference of radiation intensity between direct 125I-P-selectin and following bound by its antibody 125I-9E1 suggests that the distribution of P-selectin head’s orientation is random, with only about 10% P-selectins in their head up position. Comparingwith the traditionalmethod, thismethod is rapider,more efficient and can be widely applied to flowchamber experiment for further exploring cells rolling behavior under flows and obtaining a series of 2D molecular dynamic parameters.

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