Protein adsorption determination procedure

Also, the measured fluorescence signal may not be proportional to the surface concentration of protein. This has been observed during adsorption experiments using fluorescently-labeled 7-globulins (17) and sperm whale myoglobin (Mb) (19). Therefore, the TIRF apparatus must be properly calibrated to determine whether fluorescence intensity is indeed proportional to surface concentration. Some early calibration techniques must be viewed with caution because they were not performed under the same conditions as the protein adsorption experiments (20,21). More recently, a reliable calibration procedure that uses a double labeling technique has been developed (17). 

The unknown latex sample is injected and the centrifuge spun in the absence of flow to provide for the establishment of the particle equilibrium distribution (relaxation period). The density of the carrier solution, is determined. The flow is started and a fractogram is collected. The elution positions for the resolved components are determined and their sizes calculated corresponding to the observed retentions (no calibrations needed). The adsorption complex between a protein and one of the sample latices is formed. The above procedure is repeated using a buffer suitable for maintaining the coated particles in suspension and with an ionic strength such that particle repulsion is minimal during fractionation. The surface density of the adsorbed protein is calculated. 

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