Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation

被引:103
作者
Abas, Lindy [1 ]
Luschnig, Christian [1 ]
机构
[1] Univ Bodenkultur Wien, Univ Nat Resources & Appl Life Sci Vienna, Dept Appl Genet & Cell Biol, A-1190 Vienna, Austria
基金
奥地利科学基金会;
关键词
Microsomal fraction; Membrane protein; Arabidopsis thaliana; Endoplasmic reticulum; Plasma membrane; PIN auxin efflux carriers; ENDOPLASMIC-RETICULUM; ARABIDOPSIS-THALIANA; PLASMA-MEMBRANE; HEAT-SHOCK; PROTEINS; CELLS; AUXIN; VESICLES; VACUOLES; EFFLUX;
D O I
10.1016/j.ab.2010.02.030
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material. (C) 2010 Elsevier Inc. All rights reserved.
引用
收藏
页码:217 / 227
页数:11
相关论文
共 52 条
[1]   Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism [J].
Abas, L ;
Benjamins, R ;
Malenica, N ;
Paciorek, T ;
Wirniewska, J ;
Moulinier-Anzola, JC ;
Sieberer, T ;
Friml, J ;
Luschnig, C .
NATURE CELL BIOLOGY, 2006, 8 (03) :249-256
[2]   STRUCTURAL ORGANIZATION OF THE SPINACH ENDOPLASMIC RETICULUM-LUMINAL 70-KILODALTON HEAT-SHOCK COGNATE GENE AND EXPRESSION OF 70-KILODALTON HEAT-SHOCK GENES DURING COLD-ACCLIMATION [J].
ANDERSON, JV ;
LI, QB ;
HASKELL, DW ;
GUY, CL .
PLANT PHYSIOLOGY, 1994, 104 (04) :1359-1370
[3]   Characterization of AtSEC12 and AtSAR1 - Proteins likely involved in endoplasmic reticulum and Golgi transport [J].
BarPeled, M ;
Raikhel, NV .
PLANT PHYSIOLOGY, 1997, 114 (01) :315-324
[4]   Local, efflux-dependent auxin gradients as a common module for plant organ formation [J].
Benková, E ;
Michniewicz, M ;
Sauer, M ;
Teichmann, T ;
Seifertová, D ;
Jürgens, G ;
Friml, J .
CELL, 2003, 115 (05) :591-602
[5]  
Cherry J H, 1974, Methods Enzymol, V31, P583
[6]   The constitution of protoplasm [J].
Claude, A. .
SCIENCE, 1943, 97 (2525) :451-456
[7]  
Dallner G, 1974, Methods Enzymol, V31, P191
[8]  
de Duve C, 1971, J Cell Biol, V50, P20
[9]   THE TOBACCO LUMINAL BINDING-PROTEIN IS ENCODED BY A MULTIGENE FAMILY [J].
DENECKE, J ;
GOLDMAN, MHS ;
DEMOLDER, J ;
SEURINCK, J ;
BOTTERMAN, J .
PLANT CELL, 1991, 3 (09) :1025-1035
[10]  
Dey P.M., 1997, PLANT BIOCH, P1, DOI DOI 10.1016/B978-012214674-9/50002-3