OptiPrep™ The ideal density gradient medium for the purification of ...

BIOLOGICAL SEPARATIONSPage 2Isolation of plasma membrane form cardiac muscle by flotationUsing iodixanol gradients, isolation of a plasma membrane(PM) fraction is often achieved simultaneouslywith the purification of other subcellular membranes.Most of these methods are suitable for work with culturedcells and soft tissues such as liver but tissues suchas muscle often require a customized method. Neverthelesssome of the other iodixanol gradients may be adaptableto muscle tissue.Research into cardiac muscle function often involves thecomplex intracellular processes by which the GLUT4glucose transport protein is translocated to and from theplasma membrane (sarcolemma). Thus in fractionationsof cardiac muscle, a prime requirement is to be able resolvethe plasma membrane from GLUT-4 transportvesicles and other membrane compartments. The overallstrategy of this flotation gradient protocol is thus similarto that using a cultured cell line but the details of thetechnique are rather different.The PM is usually the least dense of all the subcellularmembranes and our application highlights a novel exploitationof this property. In this protocol, a crudeplasma membrane suspension is adjusted to a density(1.049 g/ml, equivalent to 5% w/v iodixanol) justgreater than that of the plasma membrane of cardiacmuscle, thus during the centrifugation the plasma membranewill float to the top the liquid. By layering thesample upon a small cushion of 15% (w/v) iodixanol(1.103 g/ml) and centrifuging for 16 h, a shallow gradientwill form due to diffusion of iodixanol and also tosome sedimentation of iodixanol molecules close to thebottom of the tube. This will therefore allow not onlyisolation of the plasma membrane, but also partial fractionationof some of the denser membrane compartments.Flotation often provides better recovery and/or resolutionof the least dense components of a crude fractionthan does sedimentation. Overloading the gradient(instability of the sample/gradient interface) cannot occurin a flotation format. The particular format describedin this protocol also allows the sample to becontained within a relatively large volume; this alsominimizes interactions between particles in the gradient.For detailed protocols and references see S27 on theOptiPrep Application CD or online at: www.axisshield-density-gradient-media.comIsolation of plasma membrane using a continuous gradientAlthough both continuous and discontinuous iodixanolgradients, centrifuged for 1-3 h have been used for theisolation of a plasma membrane (PM) fraction, oftensimultaneously with the purification of other subcellularmembranes, there is evidence that resolution is oftenimproved by centrifugation for longer periods, since it isonly under these conditions that true equilibrium densitybanding is achieved. In this method two strategies, employingthe longer centrifugation time format are presented.Dunphy et al and Woods et al applied the postnuclearfraction to a continuous iodixanol gradient,while Bivona et al initially used a discontinuous sucrosegradient to provide a crude plasma membrane beforeloading on to the continuous iodixanol gradient.Generally the most successful gradients for purificationof the PM cover a lower density range than do thoseprimarily aimed at resolving the Golgi and endoplasmicreticulum (ER) but because the density of the plasmamembrane (and other compartments) varies with thecell or tissue source, customized gradients may covereither a broader and/or higher density range. There ishowever another consideration in selection of thedensity range of the iodixanol gradient. Because Bivonaet al used a fraction containing principally PM and ER,they were able to use a shallow low-density gradient