GFP assay (yeast two hybrid ) Introduction The two-hybrid system exploit thi ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with DNA-binding site that regulates the expression of an adjacent reporter gene. Generally the assay is performed in yeast. It requires the construction of hybrid genes to encode: (1) a DNA-binging domain(DB) fused to a protein A (in our case:E2) (2) an activation domain(AD) fused to a protei B(in our case:ASF) The domains most commonly used are the DNA-binding domains of GAL4 and LexA, and the activation domain of Gal4 and Herpes virus VP16. One reporter gene often used is E. coli lacZ, which produces blue colonies on plates or filters containing X-gal. In addition, use of a yeast gene involved in amino acid biosynthesis ,such as HIS3 or LEU2, allows selsction for cells that grow on media lacking the revelant amino acid, and is particularly helpful. We used the GFP repoter gene instead of lacz to see if the colonoes will produce fluroscence proteins and can be visualize under UV light. Method It is known that the hinge of E2 protein interacts with the alternative splicing factor, so we set 6 reactions:
DB
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AD
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LacZ/GFP |
empty
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ASF
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E2(full-length)
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ASF
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E2กต2(truncated):hinge(RS domain) is deleted |
ASF
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ASF(Alternative splicing factor)![]()
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Result The E2-ASF set is not very distinguishable from the empty-ASF set under UV light, probably due to: (1) too late to examine the colonies under UV light; (2) the basal level transcription in empty-ASF colony Reference: The two-hybrid system: an assay for protein-protein interactions STALEY FIELD AND ROLF STERNGLANZ TIG AUGUST 1994 Vol.10 No8
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