Evaluation by Dot-Immunoassay of the Differential Distribution of Cell Surface and Intracellular Proteins in Glycosylphosphatidylinositol-Rich Plasma Membrane Domains
✍ Scribed by Subburaj Ilangumaran; Stephan Arni; Yves Chicheportiche; Anne Briol; Daniel C. Hoessli
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 156 KB
- Volume
- 235
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts Detection and isolation of plasma membrane proof lymphoma cells and intestinal tissue. The GPI-anteins using antibodies have become standard apchored proteins tend to concentrate into specialized proaches leading to their detailed structural and funcplasma membrane domains enriched in glycosphingotional characterization. Proteins in detergent lysates lipids. The dot-immunoassay has been successfully and unextracted plasma membrane fractions can be used to demonstrate the differential distribution of bound to ELISA plates (1, 2), but epitopes bound to a GPI-linked and transmembrane surface glycoproteins membrane filter support are more conveniently subof T lymphocytes in sucrose density gradient fractions of TX-100 lysate. The type II transmembrane protein jected to a variety of assays (3). Many plasma mem-CD26 and the intracellular tyrosine kinase p56 lck par-brane proteins have been detected and quantitated by tially cofractionated with GPI-linked glycoproteins, dot-immunoassays (4-12). Detergent extracts of whole and the extent to which they partition into GPI-rich cells (4-7), plasma membrane preparations (8), tissue plasma membrane domains could be evaluated. Prefhomogenates (9-11), and isolated plasma membrane erential association of the acidic glycosphingolipid vesicles (12) have been successfully screened by dot-GM1 with these domains could be demonstrated by immunoassays for cell surface proteins. The results in cholera toxin binding directly to the dot-blotted suthese studies have been obtained using three different crose density gradient fractions. Treatment of whole readouts: (i) spectrophotometric quantitation of a solucell TX-100 lysates or sucrose gradient fractions dotted ble color reaction product requiring separation of indionto nitrocellulose filter strips with bacterial phosvidual dots at the final step (4, 10-11), (ii) deposition phatidylinositol-specific phospholipase C (PI-PLC) of an insoluble color reaction product on the dot folproved to be an efficient method to assay for the preslowed by visual estimation (7) or reflectance densitomeence of a GPI-anchor in Thy-1 and Ly6 surface glycotry (9), and (iii) use of radiolabeled second antibody, proteins. We have used three criteria, namely flotation protein A, or ligand and estimation of the bound radioto light density fractions in sucrose gradients, colocalactivity by liquid scintillation counting (8) or densitoization with GM1, and sensitivity to PI-PLC cleavage, metric scanning of autoradiograms (5, 6, 12). Detection to assess the presence of a GPI modification in a putaby enhanced chemiluminescence (ECL) 2 has proved to tive GPI-linked protein in intestinal tissue extract. It is envisaged that the techniques described in this report would find a wider application to rapidly assess the 49