Charge-based separation of detergent-resistant membranes of mouse splenic B cells.
Current biochemical characterization for cholesterol- and glycolipid-rich membrane microdomains largely depends on analysis of detergent-resistant membranes (DRMs). In the present study, we succeeded in separation of DRMs of similar density-based on their electrical charge using free-flow electrophoresis (FFE). After crosslinking of B cell receptor (BCR), mouse splenic B cells were lysed with 1% Brij-58 and the resulting lysate was subjected to sucrose density gradient ultracentrifugation. The low-density fraction that recovered a part of DRMs containing IgM together with those enriched in GM1a, the Src family protein tyrosine kinase Lyn, and the alpha subunit of inhibitory heterotrimeric GTP-binding protein was further resolved by FFE. FFE separated the former into more cathodally deflected fractions than the latter. In addition, FFE revealed an anodal shift of DRMs containing a transmembrane protein CD38 upon BCR-crosslinking. The results demonstrate the effectiveness of FFE for the charge-based separation of DRMs.
Katsumata
Osamu
O
Department of Physiology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi, Matsudo, Chiba 271-8587, Japan.
Kimura
Tomoko
T
Nagatsuka
Yasuko
Y
Hirabayashi
Yoshio
Y
Sugiya
Hiroshi
H
Furuyama
Shunsuke
S
Yanagishita
Masaki
M
Hara-Yokoyama
Miki
M
D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000166:cytology*; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D008297:Male; D021962:Membrane Microdomains / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice; D013154:Spleen / Q000166:cytology* / Q000378:metabolism; D014461:Ultracentrifugation
D003902:Detergents; D008565:Membrane Proteins