Dextran retention experiment

Table 3.2 Comparison between pore radii determined by AFM and by dextran retention experiments.

This cross-axis CPC provides the universal application of protein samples with a dextran-PEG polymer-phase system. Using a prototype of the L cross-axis CPC with a 130-mL column capacity, profilin-actin complex was purified directly from a crude extract otAcanthamoeba with the same solvent system as used for the serum protein separation earlier. The sample solution was prepared by adding proper amounts of PEG 8000 and dex-tran T500 to 2.5 g of the Acanthamoeba crude extract to adjust the two-phase composition similar to that of the solvent system used for the separation. The experiment was performed by eluting the upper phase at 0.5 mL/min under a high revolution rate of 1000 rpm. The profihn-actin complex was eluted between 60 mL and 84 mL fractions and well separated from other compounds. The retention of the stationary phase was 69.0% of the total column capacity. 

A series of experiments was performed to examine the retention of the PEG-dextran polymer phase system without samples. Results are shown in Table 2, where % retention of the stationary phase is indicated at the flow rates of 0.5-1 or 2 mL/min for each spiral column. The results indicate that, among the single-spiral disks (I and II), column I with a deep channel retained over 60% of the lower dextran-rich stationary phase (U-O-H and U-0-T), at a low flow rate of 0.5 mL/min while, under other conditions, the retention was substantially less than 50% of the total column capacity. In contrast, the four-spiral disks (columns III and IV) attained remarkably im-... 

The molecular weight cut-off (MWCO) was specified by the manufacturer based on dextran experiments (see Chapter 4). The nominated pore size was estimated from the MWCO using the relation given by Wotch (1993), which is also shown in Chapter 2. The pore sizes range from 1 to 10 nm. This is based on dextran rejection and does not account for variations of retentions with molecular shape. 

Sundberg A L, Blomquist E, Carlsson J, et al. (2003). Cellular retention of radioactivity and increased radiation dose. Model experiments with EGF-dextran. Nucl. Med. Biol. 30 303-315. 

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