I1i1ii

0 40 80 120 160 200 240 [Pyrimethamine] (nM)

Fig. 6.13 Measurement of FAC data for a range of pyrimethamine concentrations applied to sol-gel-entrapped dehydrofolate reductase: (A) overlay of breakthrough curves and (B) nonlinear regression analysis of the fit to the measured breakthrough volumes from A [28]. Adapted with permission from the American Chemical Society.

suggest the constructs closely approximate immobilized systems. In any case, the sol-gel preparation is a powerful addition to conventional immobilization strategies and will serve to further shorten assay development times as well as broaden the class of proteins that can be screened by the technique. An obvious disadvantage to the entrapment method is that interaction analysis is only possible between molecular species that are widely different in hydrodynamic radius, a similar restriction experienced by screening systems based on size exclusion chromatography.

As the only requirement for conducting basic FAC experiments is the immobilization or entrapment of the target molecule in a construct that supports forced flow, many strategies for immobilization can be considered. The Wainer lab has successfully used immobilized artificial membranes as a method to retain membrane associated species and demonstrated that multiple receptors from rat brain homogenates could be successfully probed. Earlier work by Lundahl described the entrapment of liposomes and even whole cells [29]; all of these entrapment constructs can be tolerated in FAC experiments provided that forced flow does not generate excessive pressure drops across the column, potentially leading to phase collapse. In this regard, the sol-gel method shows the greatest promise of all the entrapment methods for sustaining high efficiency FAC driven by high pressure flows, but full immobilization would be required for the highest efficiency.

From this discussion, there is an obvious advantage to FAC in that the assay development approach is extremely flexible and adaptable to the requirements of the interaction to be studied. It is worth mentioning that the effort placed on creating a FAC cartridge is never wasted - it can be used as a simple capture/elute tool for alternative screening approaches, and even in preclinical studies that require methods for monitoring drug candidates and their active metabolites [30].

Was this article helpful?

0 0

Post a comment