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:  
 

  LacZ/GFP

                        
                                
                 ASF(Alternative splicing factor)        


                                
                  
                    

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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