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BioTechniques

Preparation of mammalian plasma membranes by aqueous two-phase partition.


PMID 2483665

Abstract

Plasma membranes are readily purified from crude mixtures by the technique of aqueous two-phase partition. This procedure has been used widely to prepare plasma membrane fractions, highly purified, from both green and dark-grown plant materials. Only recently, however, has the method been applied to animal cells and tissues to supplant previous protocols where preparative sucrose and other gradient procedures were employed. The method based on aqueous two-phase partition, is rapid, reproducible and facile. It is especially useful for tissue culture cells since gradient methods often are complicated by alterations in plasma membrane density with different culture conditions and the presence of extensive cytoskeleton-membrane interactions. Homogenates prepared either in dilute 1 mM bicarbonate or isotonic sucrose are first centrifuged to concentrate the plasma membrane vesicles. The concentrated membranes are then combined with a mixture of dextran and polyethylene glycol that will of itself spontaneously separate into a polyethylene glycol-rich upper phase and a dextran-rich lower phase. The mixture is usually centrifuged to accelerate phase separation. The plasma membranes enter the upper, polyethylene glycol-rich phase, whereas contaminating membranes remain with the dextran of the lower phase. The yield of plasma membranes is 20% or more of those present in homogenates and the fraction purity is 90% or greater.