Preparation of gradient solutions (mammalian)
The observed osmolality of OptiPrep™ depends on the mode of measurement (vapour pressure or freezing point); moreover the situation is complicated by the tendency of the iodixanol molecules to associate non-covalently in a concentrated aqueous solution.
Measured values for its osmolality are thus lower than might be expected. Importantly however, when OptiPrep™ is diluted with a buffered isoosmotic solution, the iodixanol oligomers dissociate and all dilutions are isoosmotic. Under normal operating conditions therefore OptiPrep™ behaves as if it had an osmolality of approx 290 mOsm.
The recommended procedure for the production of density gradient solutions or for adjustment of the density of organelle suspensions is to use a working solution (WS) whose composition is compatible with the particles to be separated.
The following methodology is based on the use of 0.25 M
sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 as homogenization medium (HM). To keep the
concentrations of EDTA and buffer constant in the gradient constant first prepare a 50% (w/v)
iodixanol working solution by mixing 5 vol. of OptiPrep™ with 1 vol. of 0.25 M sucrose, 6mM EDTA,
60 mM Tris-HCl, pH 7.4. The gradient solutions are then produced from the Working Solution (WS) by
dilution with the HM according to Table 1.