Detergent partitioning, Triton

To study partitioning ot proteins trom cells, the cells are lysed directly in the Triton/PBS mixture in the presence ot 1 mM PMSF, and, after removal of the nuclei by centrifugation, the procedure tor phase separation described above is followed. It should be noted that many antibodies do not bind well to their antigens in the presence of high concentrations ot detergent. It is therefore advisable to dilute the Triton X-114 phase with water at 0 °C before immunoabsorbtion. 

Any of the above enzymatic or chemical cleaving agents can be employed to alter the partitioning behavior of a GPI-anchored protein when extracted with Triton X-114. A Triton X-114 solution raised above 20°C separates into a detergent phase and an aqueous phase. GPI-anchored proteins will partition into the detergent phase unless released from the anchor [58] or unless some characteristies of the protein results in anomalous partitioning behavior [59-61]. 

Figure 10.17(a) The partition coefficient (P) of THC in the presence of varying concentrations of Cremophor EL. Each point is the mean of at least 15 determinations. S.E.M. values (not shown) are on average 5.4% of the mean. From Roth and Williams [98] with permission, (b) Effect of Triton X-100 on the binding of [ H]-QNB to synaptosomal membranes. The binding was carried out with 0.9 nM [ H]-QNB in the presence of various concentrations of Triton X-100. The 100 % represents the specific radioactivity of controls without detergent (i.e., 1.73 pmol/mg protein). The points are the means of triplicate determinations. From Aguilar et al [100] with permission. 

All the samples are subjected to gel electrophoresis and Western blot analysis. The integral membrane protein neurexin was found to be highly enriched in the membrane extract and resistant to alkaline wash (Figure 32.3). Nonionic detergents, e.g., NP-40 and Triton, released the protein from the membrane. The protein partitioned into the detergent phase after Triton X-114 cloudy-point precipitation. 

Similarly, incorporation of monomeric phospholipids by detergents into mixed micelles results in a chemical-shift change in P NMR, and this can be used to follow the micellization process. This is illustrated in Fig. 8, where the micellization of dihexanoyl-PC by titration with Triton X-lOO was followed by changes in the P-NMR chemical shift. In contrast, with the same amount of Triton X-100, dibutyryl-PC is hardly micellized at all. A control titration of the completely water-soluble analog glycerophosphoryl-choline is also included. For the dihexanoyl-PC, a partition coefficient between Triton X-100 micelles and free solution was calculated as a function of the Triton X-100 concentration, using the phase-separation approximation for mixed-micelle formation and considering the mixed micelles to be a pseudo-phase. With the data in Fig. 8, the fraction of dihexanoyl-PC... 

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