High throughput assay systems and methods for identifying agents that alter expression of cellular proteins

摘要

595791 Disclosed is a method of identifying an agent that alters the level of surface expression of a protein in a mammalian cell, said method comprising: a) preparing a first medium containing mammalian cells that express said protein; b) adding to said first medium containing mammalian cells an effective amount of a candidate agent; c) incubating said cells in the presence of said candidate agent for a sufficient period of time; d) treating said cells with an effective amount of a fixative; e) adding to said first medium containing mammalian cells an effective amount of at least one primary antibody followed by an effective amount of at least one secondary antibody, wherein said primary antibody binds to at least one extracellular epitope of said protein and said secondary antibody binds to said first antibody, and further wherein said extraceullular epitope contains a tag; and f) determining the level of binding of said primary antibody to said protein with said candidate agent, wherein a change in said level of binding relative to control indicates that said candidate agent alters the level of surface expression of said protein. Further disclosed is a method of identifying an agent that alters the level of expression of a protein in a mammalian cell, said method comprising: a) preparing a frrst medium containing mammalian cells that express said protein; b) adding to said frrst medium containing mammalian cells an effective amount of a candidate agent; c) incubating said cells in the presence of said candidate agent for a sufficient period of time; d) treating said cells with a fixative followed by a permeabilizing agent; e) adding to said frrst medium containing mammalian cells an effective amount of at least one primary antibody followed by an effective amount of at least one secondary antibody, wherein said primary antibody binds to at least one extracellular epitope of said protein and said secondary antibody binds to said frrst antibody, and further wherein said extraceullular epitope contains a tag; and f) determining the level of binding of said primary antibody to said protein with said candidate agent, wherein a change in said level of binding relative to control indicates that said candidate agent alters the level of expression of said protein. Further disclosed is a method of identifying a peptide that alters the level of expression of an integral membrane protein in a mammalian cell, said method comprising: a) preparing a fIrst medium containing mammalian cells that express said protein; b) adding to said frrst medium containing mammalian cells a retroviral expression library; c) adding to said frrst medium containing mammalian cells an effective amount of at least one primary antibody that binds to at least one extracellular epitope of said protein; d) adding to said medium a fluorescently tagged secondary antibody; and e) sorting said mammalian cells based on fluorescence for an increase in cell surface expression and identifying peptides responsible for said increased expression; wherein said at least one extracellular epitope contains a tag.