title: Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium.
abstract: We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H+L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10(-11) to 8.1 x 10(-10) mol L(-1).
authors.affiliation: School of Chemistry and Chemical Engineering, Shandong University, Jinan, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Guang
authors.initials: G
authors.lastname: Xu
authors.forename: Zhikun
authors.initials: Z
authors.lastname: Shi
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.forename: Wenrui
authors.initials: W
authors.lastname: Jin
mesh_terms: D000327:Adsorption; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000032:analysis*
chemical_list: D007143:Immunoglobulin Heavy Chains; D011506:Proteins