Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping.
Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.
Department of Plant Biology and Biotechnology, University of Copenhagen, Copenhagen, Denmark.
Anette
A
Lund
Anja Thoe
AT
Fuglsang
D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D017136:Ion Transport; D008861:Microsomes; D059828:Plant Cells / Q000737:chemistry* / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry; D017494:Proton Pumps / Q000502:physiology*; D012996:Solutions; D013347:Subcellular Fractions
D011108:Polymers; D017494:Proton Pumps; D012996:Solutions