Frequently, a simple post-nuclear supernatant is used as a gradient input rather than a partially purified fraction such as a light mitochondrial pellet or microsomes. Since the gradient is often used not so much to purify one particular type of membrane (see Chapter 4), but rather to analyse the translocation of proteins or other molecules from compartment to compartment, it is often considered more important that none of the potentially involved compartments gets lost in some pre-gradient treatment. Such a simple treatment of the homogenate also reduces the experimentation time and number of manipulations to a minimum.
For many years, sucrose gradients were the principal vehicle for analysing these membrane compartments and a number of the most well-established protocols using this solute are presented in this chapter. Increasingly, however, the use of iodinated density gradient media, notably Nycodenz® and iodixanol, are being used for this purpose and a number of examples of the use of these gradient solutes are given. They have several advantages over sucrose; they are commercially available as dense solutions, making gradient solutions easy to prepare, and their lower osmolality compared to sucrose solutions of similar density (particularly iodixanol solutions) often results in a greater resolving power than that of sucrose.
The viscosity of Nycodenz® and iodixanol solutions is also lower than that of sucrose solutions of similar density and there are many examples that take advantage of the consequent more rapid movement of membrane particles through the gradient. Gradients of Nycodenz® and iodixanol are frequently run for 2-4 h, rather than overnight, which
is the norm for sucrose gradients. However, there is a school of thought that maintains that to get true equilibrium density banding it is necessary to centrifuge for long periods (> 12 h) at relatively low centrifugation speeds, irrespective of the gradient medium. A number of iodixanol gradient methods support this contention (see Protocol 5.8). There are also some examples of the use of very short centrifugation times, which tend to separate particles principally on the basis of sedimentation velocity (see Protocol 5. 9).
One property of iodixanol that is not available for sucrose is its ability to form self-generated gradients, given a sufficiently high g-force and the right sort of rotor (vertical or near-vertical) . There are several advantages of using self-generated gradients over the more standard preformed gradients: they are very easy to prepare (the sample is simply adjusted to a certain iodixanol concentration); they are very reproducible and very shallow gradients may be generated (under the appropriate centrifugation conditions) - such gradients are not easily preformed. Protocol 5.15 is an example of the use a self-generated gradient.
Thus, the protocols in this chapter are generally not aimed at the isolation of an individual membrane compartment; rather they describe the use of a variety of gradient systems that might be used for analysing some aspect of membrane trafficking or cell signalling. An exception is the group of protocols at the start of this chapter that describes the isolation of various types of plasma membrane domain. Because plasma membrane domains have a unique composition and function, they are of particular importance in studies on the means by which the cell directs and controls the flow of molecules to and from the surface.
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