Patent ID: 6632924
Filing Date: 2003-10-14
Classification: C07K,C12N

Abstract:
A method of assessing whether a condition or a stimulus causes translocation of a protein of interest from an intracellular location to the plasma membrane in mammalian cells, comprising:(a) culturing mammalian cells expressing a modified protein of interest under a condition or a stimulus to be assessed for its effects on translocation of the protein of interest, wherein the modified protein of interest comprises the protein of interest and at least one epitope tag in an extracellular domain and a fluorescent tag in an intracellular domain thereof, wherein the cells are referred to as test cells; (b) detecting the fluorescent tag, which is indicative of total modified protein of interest present in the test cells, and the epitope tag, which is indicative of modified protein of interest present at the cell membrane in the test cells; (c) determining a proportion of the modified protein of interest at the cell membrane in the test cells of (b) to the total modified protein of interest in the test cells of (b), thereby producing a test value; and (d) comparing the test value with a control value, wherein the control value corresponds to a proportion of modified protein of interest at the cell membrane in control cells to total modified protein of interest in control cells, and the control value is determined from control cells which are the same cells as are cultured in (a) and which are cultured under the same conditions as in (a), except that the control cells are not cultured under the condition or stimulus to be assessed, wherein if the test value is greater than the control value, then the condition or stimulus causes translocation of the modified protein of interest from an intracellular location to the plasma membrane in mammalian cells.