A fast and sensitive method for isolation of detergent-resistant membranes from T cells.
We describe a fast and sensitive method for isolation of detergent-resistant membranes (DRMs) from T cells by sucrose density gradient centrifugation using a smaller accumulated centrifugal force in a tabletop ultracentrifuge. Compared to previous reports, this method, which requires less biological material, is faster and permits quantitative separation of DRMs from other cellular membranes with good resolution. The method, which can be completed in 6 h, yields more than 80% of the total content of DRM-associated adaptor molecules LAT (linker for T cell activation), PAG/Cbp (protein associated with glycosphingolipid-enriched microdomains or Csk-binding protein) and LIME (Lck-interacting membrane protein) in low-density fractions using only 2x10(7) T cells.
Lygren
Birgitte
B
Biotechnology Centre of Oslo, University of Oslo, Norway.
Taskén
Kjetil
K
Carlson
Cathrine R
CR
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient / Q000379:methods*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000737:chemistry*; D014461:Ultracentrifugation / Q000379:methods*
D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008565:Membrane Proteins; C498710:PAG1 protein, human; D010750:Phosphoproteins