Methods For The Detection, Analysis And Isolation of Nascent Proteins

Abstract

This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.

1 . A composition, comprising a nascent protein bound to a surface, wherein said nascent protein comprises an N-terminal epitope marker and a C-terminal epitope marker incorporated into said nascent protein during translation, said N-terminal epitope marker bound to a first binding agent and said C-terminal epitope marker bound to a second binding agent. 
     
     
         2 . The composition of  claim 1 , wherein said surface is a well of a microtiter plate. 
     
     
         3 . The composition of  claim 1 , wherein said nascent protein is encoded by a gene associated with a disease. 
     
     
         4 . The composition of  claim 1 , wherein said surface comprises a bead surface. 
     
     
         5 . The composition of  claim 1 , wherein said surface comprises a microfluidic device. 
     
     
         6 . A composition, comprising a nascent protein bound to a surface, wherein said nascent protein comprises an N-terminal epitope marker and a C-terminal epitope marker incorporated into said nascent protein during translation, said N-terminal epitope marker bound to a first antibody and said C-terminal epitope marker bound to a second antibody. 
     
     
         7 . The composition of  claim 6 , wherein said surface is a well of a microtiter plate. 
     
     
         8 . The composition of  claim 6 , wherein said nascent protein is encoded by a gene associated with a disease. 
     
     
         9 . The composition of  claim 6 , wherein said surface comprises a bead surface. 
     
     
         10 . The composition of  claim 6 , wherein said surface comprises a microfluidic device. 
     
     
         11 . A microfluidic device comprising fluid, said fluid comprising a nascent protein, wherein said nascent protein comprises an N-terminal epitope marker and a C-terminal epitope marker incorporated into said nascent protein during translation. 
     
     
         12 . The microfluidic device of  claim 11 , wherein said device comprises channels. 
     
     
         13 . The microfluidic device of  claim 12 , wherein said channels are between approximately 0.10 and 0.50 micrometers in depth. 
     
     
         14 . A method, comprising: a) providing a microfluidic device and reagents for translating a nascent protein, and b) translating a nascent protein in said microfluidic device, wherein said nascent protein comprises an N-terminal epitope marker and a C-terminal epitope marker incorporated into said nascent protein during translation. 
     
     
         15 . The method of  claim 14 , wherein said device comprises channels. 
     
     
         16 . The method of  claim 15 , wherein said channels are between approximately 0.10 and 0.50 micrometers in depth.