A novel cell-based scintillation proximity assay for studying protein function and activity in vitro using membrane-soluble scintillants.
Here we describe for the first time a cell-based scintillation proximity assay using membrane soluble scintillants (MSS). MSS have a scintillant "head" group (2,5-diphenyloxazole) attached to a lipophilic "tail." MSS do not scintillate in an aqueous environment in the presence of a radioactive source: however, in a non-aqueous environment, such as a lipid bilayer (e.g., liposome or cell membrane), scintillation does occur. MSS can be incorporated into liposomes. When these MSS-containing liposomes are fused with the plasma membranes of cells in culture the MSS are incorporated into the cell membrane. Radiolabelled molecules in close proximity to the cell membrane will then elicit a scintillation signal. This system has been used to successfully monitor [(14)C]methionine uptake in HeLa cells and may be used in radiochemical and radioligand binding assays either in vivo or on microsomal preparations obtained from tissues. This new scintillation proximity technology could be readily adapted for high-throughput screening.
Sense Proteomic Ltd., Babraham Hall, Babraham, Cambridge CB2 4AT, UK. info@senseproteomic.com
Steven J
SJ
Culliford
Patrick
P
McCauley
Andrew J
AJ
Sutherland
Mark
M
McCairn
John
J
Sutherland
Jonathan
J
Blackburn
Roland Z
RZ
Kozlowski
D001671:Biochemistry / Q000295:instrumentation* / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012588:Scintillation Counting; D012997:Solvents; D013997:Time Factors
D008081:Liposomes; D010713:Phosphatidylcholines; D011506:Proteins; D012997:Solvents; D008715:Methionine