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Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption

1983; Elsevier BV; Volume: 150; Issue: 1-2 Linguagem: Inglês

10.1016/s0022-0728(83)80209-5

2590-2954

Steven A. Rockhold, Roswell Quinn, R. A. Van Wagenen, Joseph D. Andrade, Manuela Reichert,

Advanced Biosensing Techniques and Applications

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.