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

AXIS-SHIELDISSUE 10, 2009BIOLOGICAL SEPARATIONSNEWS BULLETIN FOR AXIS-SHIELD DENSITY GRADIENT MEDIAApplications• Isolation of PM fromcardiac muscle byflotation• Isolation of PM anddomains using cationiccolloidal silica• Isolation of PM byflotation through acontinuous gradient(16h spin)• Isolation of PM usinga discontinuous gradient• Isolation of PM bysedimentationthrough a pre-formedgradient (1-3h spin)• Detergent-free purificationof caveolae• Purification of detergent-resistantlipidrafts• Purification of lipidrafts in the absenceof detergentOptiPrepThe ideal density gradient medium for thepurification of plasma membrane and domainsOptiPrep is a sterile endotoxintested solution of 60% iodixanol in waterwith a density of 1.32 g/ml.Iodixanol was developed as an X-raycontrast medium an has therefore beensubjected to rigorous clinical testing.Iodixanol is non-ionic, non-toxic tocells and metabolically inert.Iodixanol solutions can be made isoosmoticat all useful densities.Iodixanol solutions have low viscosityand osmolarity.OptiPrep is manufactured in compliancewith current EU guide to cGMP.Actual endotoxin levels in each batchare usually measured at < 0.13 EU/ml.OptiPrep offers three gradient formatsfor the isolation of the plasmamembrane (PM).Continuous or discontinuous gradients(1-3h)Continuous gradients (12-16h)Flotation through discontinuous gradientsOptiPrep offers two approaches tothe purification of lipid-rich micro domains.The first to be developed involves theisolation of caveolae from a sonicatedtotal plasma membrane fraction. Thisstrategy has also been used for the isolationof lipid rafts.The lipid-rich nature of these microdomainsrenders them relatively insensitiveto detergent solubilisation, comparedto the rest of the plasma membraneand this forms the basis of theirseparation in detergent containing iodixanolgradients.

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

ISSUE 10, 2009Page 3(5-20% (w/v) iodixanol. A total post-nuclear fractionhowever also contains organelles such as mitochondriaand peroxisomes which are much more rapidly sedimentingthan the small vesicles of the PM and ER andmay disturb such shallow low-density gradients. ThusDunphy et al and Woods et al chose steeper gradientscovering a wider density range.For a detailed protocol and references see S25 on theOptiPrep Application CD or online at www.axisshield-density-gradient-media.comDetergent free purification of caveolae by sonication of a PM fractionThis is a detergent-free strategy and was first describedby Smart et al for the isolation of a caveolae from humanfibroblasts. It was later modified by Babitt et aland subsequently it has been applied either in the originalor the revised form (without any further adaptation)to a wide range of other cell types and to membranefractions from brain tissue, the parathyroid gland andchicken intestinal mucosal epithelium.10-20%23%5%15%~25%According to all published papers the sonicated plasmamembrane suspension is adjusted to 23% iodixanol andlayered beneath a continuous 10-20% iodixanol gradientand centrifuged at 52,000g for 90 min. After harvestingthe gradient (from the top) in 15 fractions morethan 95% of the protein was detected in fractions 9-15while the caveolin was more or less confined to fractions3-12. In the top 7 or 8 fractions therefore the specificactivity of the caveolin is very high and it is thiscut of the gradient which is processed further in theconcentration phase.Approximately the top half of the gradient is readjustedto 23% iodixanol; in the original method this was then52,000g/90 min52,000g/90 minoverlaid with a solution of 5% iodixanol and the centrifugationrepeated to allow the caveolae to bandsharply at the interface. Subsequently a layer of 15%iodixanol was interposed to remove any denser proteinrichmaterial harvested from the first gradient (see figure).Some of the published papers report the use of theoriginal second gradient, some use the modification.For a detailed protocol and references see S35 on theOptiPrep Application CD or online at www.axisshield-density-gradient-media.comPurification of lipid rafts as detergent-resistant membranes /DRMs)In one of the most widely used strategies, lipid raft isolationis based on the insolubility of these domains in anon-ionic detergent such as Triton X100. Detergent isadded either to intact cells or a postnuclear supernatant,which is then normally adjusted to a high density andlayered under a discontinuous gradient. Lipid rafts,which have a relatively low density, float away fromsoluble proteins and dense cytoskeleton- associatedproteins, which remain in the load zone. Originally sucrosegradients were used for the separation but whenNycodenz® flotation gradients were introducedNaslavsky et al observed that these gradients were farsuperior to those of sucrose.Routinely cell lysis or homogenisation is performed in abuffered saline containing a chelating agent, a detergent,

Web:www.axis-shield-density-gradientmedia.comAXIS-SHIELDIn this leaflet we have presented some of the applicationsavailable for the isolation of plasma membrane anddomains using OptiPrep. More information can befound at: www.axis-shield-density-gradientmedia.com.On the front page click on “Subcellularmembranes and cell organelles”.PO Box 6863 RodelokkaN-0504 OsloNorwayPhone: +47 24 05 60 00Fax: +47 24 05 60 10Email: bjh@no.axis-shield.com orjohn@jgrescon.fsbusiness.co.ukprotease inhibitors and/or other additives such as DTTor CaCl 2 . Whether the light mitochondrial fraction isused to prepare Golgi membranes depends on their responseto homogenization; if they vesiculate they willbe recovered in the microsomal fraction; if they retain atubular structure they will be recovered in the light mitochondrialfraction. They are the least dense membranein the light mitochondrial fraction and can be effectivelyisolated in a self-generated OptiPrep gradient(see figure).Pre-formed gradient may also be used. Note that theisolation of Golgi membrane vesicles and other componentsof the secretory endocytic and synthetic systemsare described elsewhere.Lyse cells in buffer+ detergent± liquid shearDRM150,000-180,000g4 hDisrupt cells in bufferby liquid shearAdd detergentBuffer20%30%35%Disrupt cells in bufferby liquid shear1000-3000g/5 minSupernatantAdd detergentMaintain at 4 o C for 30 minAdjust to 40% iodixanolFor a detailed protocol and references see S33 on theOptiPrep Application CD or online at: www.axisshield-density-gradient-media.com