Rotofor cell

When the acidic (A) and basic (B) components of a particular buffer pair are mixed in the proportions shown (80%A 20%B 50%A 50%B or 20%A 80%B) and used for pH control in the Rotofor cell, the tabulated approximate pH gradients will be generated. Other ratios of the A and B components can also be used. See Refs. 24 and 25. 

When RotoLyte buffer pairs are used to establish the pH, the operation of the Rotofor cell is the same as with carrier ampholytes. The choice of the proper buffer pair requires that the pi of the protein of interest be known. The sample is diluted with water to half the chamber volume and then mixed with an equal volume of the appropriate blend of buffers (Table 2). Refractionation is usually not required with buffer pairs. A single run often gives the protein of interest in the desired degree of purity. 

The ideal sample run on the Rotofor cell would contain only the protein mixture, water, and ampholytes or buffers. However, pi precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12°C. Detergents (1-2% w/v) may also be added to samples. Zwitterionic detergents, such as CHAPS, CHAPSO, and nonionic octyl-glucoside are satisfactory. 

For preparative lEF, the Rotofor cell (Bio-Rad) can be used. Here, a column is used for focusing. Convection streams are inhibited by a row of inserted filters and the rotation of the column around the horizontal axis. The Rotofor cell was developed by Egen et al. 1984. 

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