Preparative two-dimensional gel electrophoresis of membrane proteins

被引:112
作者
Pasquali, C [1 ]
Fialka, I [1 ]
Huber, LA [1 ]
机构
[1] Res Inst Mol Pathol, IMP, A-1030 Vienna, Austria
关键词
membrane protein separation; sample application; in-gel reswelling; preparative two-dimensional polyacrylamide gel electrophoresis; epithelial cell line;
D O I
10.1002/elps.1150181413
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Electrophoretic techniques, and especially two-dimensional gel electrophoresis (2-DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well es for the identification of protein-protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first cr second dimension of 2-DE. Here we describe the modification of a protocol using a combination of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4-dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver-stained gels obtained from identical samples (4 mu g). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale-up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialka et al., Electrophoresis 1997, 18, 2582-2590).
引用
收藏
页码:2573 / 2581
页数:9
相关论文
共 37 条
[1]   Two-dimensional electrophoresis of membrane proteins: A current challenge for immobilized pH gradients [J].
Adessi, C ;
Miege, C ;
Albrieux, C ;
Rabilloud, T .
ELECTROPHORESIS, 1997, 18 (01) :127-135
[2]  
APPEL R, 1985, COMPUTER GENERATED I, V4, P118
[3]  
APPEL RD, 1997, IN PRESS ELECTROPHOR, V18
[4]   ISOELECTRIC-FOCUSING IN IMMOBILIZED PH GRADIENTS - PRINCIPLE, METHODOLOGY AND SOME APPLICATIONS [J].
BJELLQVIST, B ;
EK, K ;
RIGHETTI, PG ;
GIANAZZA, E ;
GORG, A ;
WESTERMEIER, R ;
POSTEL, W .
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, 1982, 6 (04) :317-339
[5]   A NONLINEAR WIDE-RANGE IMMOBILIZED PH GRADIENT FOR 2-DIMENSIONAL ELECTROPHORESIS AND ITS DEFINITION IN A RELEVANT PH SCALE [J].
BJELLQVIST, B ;
PASQUALI, C ;
RAVIER, F ;
SANCHEZ, JC ;
HOCHSTRASSER, D .
ELECTROPHORESIS, 1993, 14 (12) :1357-1365
[6]   ENDOCYTOSIS IN FILTER-GROWN MADIN-DARBY CANINE KIDNEY-CELLS [J].
BOMSEL, M ;
PRYDZ, K ;
PARTON, RG ;
GRUENBERG, J ;
SIMONS, K .
JOURNAL OF CELL BIOLOGY, 1989, 109 (06) :3243-3258
[7]   Subcellular fractionation of polarized epithelial cells and identification of organelle-specific proteins by two-dimensional gel electrophoresis [J].
Fialka, I ;
Pasquali, C ;
Lottspeich, F ;
Ahorn, H ;
Huber, LA .
ELECTROPHORESIS, 1997, 18 (14) :2582-2590
[8]   The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions [J].
Fialka, I ;
Schwarz, H ;
Reichmann, E ;
Oft, M ;
Busslinger, M ;
Beug, H .
JOURNAL OF CELL BIOLOGY, 1996, 132 (06) :1115-1132
[9]   ISOLATION OF INTRACELLULAR MEMBRANES BY MEANS OF SODIUM-CARBONATE TREATMENT - APPLICATION TO ENDOPLASMIC-RETICULUM [J].
FUJIKI, Y ;
HUBBARD, AL ;
FOWLER, S ;
LAZAROW, PB .
JOURNAL OF CELL BIOLOGY, 1982, 93 (01) :97-102
[10]   ELIMINATION OF POINT STREAKING ON SILVER STAINED TWO-DIMENSIONAL GELS BY ADDITION OF IODOACETAMIDE TO THE EQUILIBRATION BUFFER [J].
GORG, A ;
POSTEL, W ;
WESER, J ;
GUNTHER, S ;
STRAHLER, JR ;
HANASH, SM ;
SOMERLOT, L .
ELECTROPHORESIS, 1987, 8 (02) :122-124