A homogeneous cell-based assay to measure nuclear translocation using beta-galactosidase enzyme fragment complementation.
Positional complementation describes the use of homogeneous assays using beta- galactosidase (beta gal) enzyme fragment complementation to detect cellular protein translocation. This phenomenon occurs when the protein of interest, recombinantly expressed as a fusion protein with a modified alpha fragment of beta gal, translocates to a cellular compartment expressing an enzyme acceptor fragment of the enzyme. When these fragments interact, high-affinity complementation occurs, and a signal is generated that is then detected upon cell lysis. In the present paper the use of positional complementation is exemplified by measuring nuclear translocation of the glucocorticoid receptor in Chinese hamster ovary-K1 cells. The approach thus provides for homogeneous protocols, in an endpoint microtiter plate assay format, without the use of either imaging or reporter gene techniques. Consequently, these characteristics suggest that the technique is suitable for automated instrumentation protocols used in high throughput screening campaigns designed to identify activators or inhibitors of nuclear translocation.
Fung
P
P
DiscoveRx Corp., Fremont, CA 94538, USA.
Peng
K
K
Kobel
P
P
Dotimas
H
H
Kauffman
L
L
Olson
K
K
Eglen
R M
RM
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008856:Microscopy, Fluorescence / Q000379:methods*; D010446:Peptide Fragments / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase