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One- and Two-Hybrid Systems

GNGK03 - Grow'n'Glow GFP One-Hybrid System

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The Grow'n'Glow GFP One-Hybrid kit isolates genes for proteins that bind a specific DNA element of interest. This one-hybrid system can be used to investigate the bases and amino acids involved in specific DNA-protein interactions. The Grow'n'Glow system offers maximum sensitivity because detection of the DNA-protein interaction occurs in vivo, where proteins are more likely to be in their native conformation.

Use of the Green Fluorescent Protein (GFP) reporter eliminates the need for time-consuming beta-galactosidase lift assays. The GFP variant expressed by the GFP reporter plasmid, GFPuv, has the same excitation and emission maxima as wild-type GFP, but is 18 times brighter than the wild-type variant. Positive colonies with DNA-binding proteins brightly glow green. The glowing colonies are easily detected by placing the plate with the yeast colonies on a standard 300 nm UV transilluminator or under a UV lamp in a darkroom.

Expression of fusion proteins by the prey plasmid is controlled by the GAL1 upstream-activation sequence. In yeast with an intact galactose regulatory system, the GAL1 activation sequence is induced by galactose and repressed by glucose. This regulation inhibits expression of library/activation domain fusions until the actual screening, which prevents any potentially toxic library proteins from affecting growth of the yeast. These toxic proteins would be missed during screening in a non-inducible system.

The One-Hybrid Approach with Grow'n'Glow.

Features

  • Offers maximum sensitivity because protein interactions occur in vivo
  • Positive colonies brightly fluoresce green and can easily be seen under UV light
  • Does not require time-consuming beta-galactosidase lift assays 
  • Finds potentially toxic proteins by screening with galactose-inducible expression libraries

Applications

  • Isolating genes for novel DNA-binding proteins
  • Mapping residues and regions responsible for DNA-binding

Contents

  • pGNG2, lyophilized DNA, 5 µg
  • pJG4-5, lyophilized DNA, 5 µg
  • GNGprimer, 500 pmol
  • 5'-PREYprimer (0.1 nmol/µl), 500 pmol
  • 3'-PREYprimer (0.1 nmol/µl), 500 pmol
  • Yeast strain EGY48, glycerol stock, 1ml
  • Prey control plasmid pJG4-5-p53, 5 µg
  • GFP positive control vector pGNG2-p53, 5 µg

Literature