A fast, simple and sensitive method for the detection and quantification of detergent-resistant membranes.
The aggregation of the T cell receptor and other signaling molecules leads to the formation of large molecular activation clusters in the cell membrane. These molecular clusters are associated with a high concentration of cholesterol, sphingomyelin and gangliosides and were referred to as lipid microdomains. Electron microscopy studies of viable cells indicate that distinct subgroups of lipid microdomains exist and they contain different types of signaling molecules. Lipid microdomains are insoluble in ice-cold solutions containing detergent and are also referred to as detergent-resistant membranes (DRM). Currently, sucrose density centrifugation is the standard method for DRM isolation. Cholera toxin B subunit (CTB) is a specific ligand for ganglioside GM1 and can be used for the detection of GM1 containing DRM. In this paper, we describe a new and simple method for quantification of GM1 associated with DRM. We used a CTB-horseradish peroxidase (HRP) conjugate and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) for the detection of DRM in floating fractions of a sucrose density gradient. Absorbance values (A(405)) were determined using a microtiter plate and an ELISA plate reader. The linear range for the HRP-ABTS reaction was determined in the presence of lysis buffer and sucrose concentrations up to 40%. Linearity of the assay was determined over a wide range (5-1000 microU peroxidase activity per well) in a single experiment and the limit of detection of this method was approximately 10 ng of CTB per gradient fraction. The method is nonradioactive, rapid and easy and can be used for the analysis of DRM resident proteins. We applied this method to Jurkat T cells and after centrifugation observed the existence of DRM floating to 5%/30% sucrose interface. After separation of the sucrose gradient, we identified a large GM1 content in the corresponding fractions 4 to 6. The presence of protein in these fractions was confirmed by silver-stained polyacrylamide gels. We confirmed the presence of adaptor molecules (LAT) and Src kinases (Lck) in the DRM containing fractions 4 to 6.
Blank
Norbert
N
Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nuremberg, Glueckstrasse 4A, Germany. Norbert.Blank@med3.imed.uni-erlangen.de
Gabler
Christoph
C
Schiller
Martin
M
Kriegel
Martin
M
Kalden
Joachim R
JR
Lorenz
Hanns-Martin
HM
D052160:Benzothiazoles; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient / Q000379:methods; D002772:Cholera Toxin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005677:G(M1) Ganglioside / Q000032:analysis*; D006801:Humans; D019169:Jurkat Cells; D008563:Membrane Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D017830:Octoxynol / Q000737:chemistry; D012680:Sensitivity and Specificity; D016622:Silver Staining; D013395:Sucrose / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry
D052160:Benzothiazoles; D008563:Membrane Lipids; D008565:Membrane Proteins; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D005677:G(M1) Ganglioside; D013395:Sucrose; D017830:Octoxynol; D002772:Cholera Toxin