Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption
Total internal reflection fluorescence (TIRF) is a powerful new method for probing protein adsorption at solid-liquid interfaces. A quantitation method for determining the amount of adsorbed protein on hydrophilic glass and quartz surfaces has been developed. This method utilizes fluorescent, non-adsorbing dextran as a calibration molecule which approximates the diffusive properties of the protein under study. Through the use of conventional bulk solution fluorescence of both protein and calibration molecules in conjunction with a TIRF calibration, graphical quantitation of the surface concentration of adsorbed protein can be performed. quantitative results can be obtained either via a graphical procedure using fluorescence calibration standards or by a numerical method which accounts for the characteristics of the decaying evanescent, interfacial wave. The graphical quantitation procedure is outlined and demonstrated with data from five gamma-globulin adsorption experiments on hydrophilic glass and quartz surfaces. The results generated by this quantitation procedure are similar to values derived from the numerical quantitation calculation. © 1983 Elsevier Sequoia S.A.
Rockhold, SA; Quinn, RD; Wagenen, RAV; Andrade, JD; Reichert, M
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