The procedure, excluding the preparation of the plasma membrane, will take approx. 4.5 h.

(© Metal 'filling' cannulas (i.d. 0.81.0 mm) can be obtained from any surgical equipment supplies company.

©2 The method used to prepare the plasma membrane will vary with the type of tissue or cell. As the source material may be a 'routine' cultured cell line such as human fibroblasts [54,55], COS 7 [56,57] or NIH 3T3 [18], a genetically modified line such as CHO-ldlA-7 [58,59] or tissue-derived cells such as lung microvascular endothelial cells [60,61] or brain astrocytes [62], the operator should refer to the appropriate literature for a plasma membrane isolation technique. However, one of the methods described in Protocols 5.8 and 5.9 may be relevant.

©3 If the sonicator does not conform to these parameters, it may be necessary to modulate the conditions in order to achieve the necessary fragmentation of the membrane. This can only be monitored by determining the caveolin and protein distribution in the first gradient (step 9).

©4 The volume of the gradient needs to be calculated to fill the tube when the 4 ml sample is underlayered. An approximately 9 ml gradient was used by Smart et al. [17]. The method can be scaled up or down as required, adjusting the volumes of sample and gradient proportionately.

©5 If a gradient-forming device is not available, then prepare a discontinuous gradient from equal volumes of 10, 15 and 20% iodixanol and allow them to diffuse overnight at 4 °C. See ref. 26 for more information about making gradients.

©6 If the volume of the gradient is increased, the volume occupied by the caveolae-containing fractions may also increase. As an approximation, the top third-to-half of the gradient should be removed. If the relative volumes of gradient and sample are significantly different it may be advisable to check the distribution of the cave-olae by assaying fractions for cave-olin by electroblotting (see Protocols 5.10-5.12).

© Until the banding of the caveolin-rich, protein-poor material in the gradient is well established, it might be pertinent to collect the gradient by upward displacement with a dense medium; for more information on unloading gradients see ref. 26. Once the banding position of the caveolae has been established, the relevant part of the gradient can be removed using a syringe and cannula.

©8 The volumes used in the second gradient may also have to be modified for larger volume tubes; it is probably good practice to increase the volumes of the sample and the two iodixanol layers, proportionally.

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