Produkty
Producenci

Grow 'n' Glow Two-Hybrid System "Complete Kit"

nr kat.: GNGK01
Opakowanie: zestaw
Cena brutto: do wyceny
ilość szt.

towar niedostępny

dodaj do przechowalni

Opis

Advantages of the Grow'n'Glow Two-Hybrid System
• protein-protein interaction is detected directly by visualisation under UV light
• greatly reduces amount of time & effort needed to screen a cDNA library
• allows immediate inspection of yeast colonies for gene activation
• non-invasive, chemical-free and cost-free assay
• GFP may be used as sole reporter gene
• higher threshold for detection of protein-protein interactions
• can be integrated simply into pre-existing LexA bait-based two-hybrid systems
• stable GFP expression
• using prokaryotic (LexA and B42) rather than eukaryotic (GAL4) proteins reduces number of false positives
• inducible expression of library fusion proteins (with galactose) reduces potential toxicity problems
• antibodies to HA-tag (fused downstream of B42) permit simple coimmunoprecipitation assay of bait and potential positive
• yeast strains with varying LEU2 sensitivities reduce problems associated with bait autoactivation

General
The yeast two-hybrid system1 or interaction trap2 has rapidly become a widely used technique in molecular biology. It is now the method of choice to identify protein-protein interactions from either cDNA libraries or known gene sequences3. The method relies on the transactivation of reporter genes in Saccharomyces cerevisiae to identify positive interactions. Growth selection via activation of either a HIS3 or LEU2 reporter gene is used in conjunction with a second reporter gene, such as LacZ, which expresses β- galactosidase. The use of two reporter genes enables discrimination of false positives which activates only the auxotrophic marker.

Yeast colonies that survive the growth selection scheme are chosen, re-streaked and tested for expression of the LacZ gene by means of a filter assay or growth on minimal medium plates containing 5-bromo-4-chloro- 3-indolyl-β-D-galactopyranoside (X-Gal). This latter step can often be time-consuming depending on the number of primary transformants obtained.
In order to simplify and accelerate the screening process of the two-hybrid systems, the new reporter plasmid pGNG14 was constructed that utilizes the green fluorescent protein (GFP) from the bioluminescent cnidarian Aequorea victoria5.

Background
The Grow'n'Glow system is a LexA-based version of the yeast two-hybrid system originally developed by Fields and Song1. The yeast two-hybrid system has proven to be a powerful tool for identifying proteins from an expression library which can interact with one's protein of interest. The technology is based on the fact that many eukaryotic transcriptional activators, such as GAL4, consist of two physically separable modular domains, one DNA-binding domain and one transcriptional activation domain. In order to activate transcription, they have to be physically tethered to one another, and neither of them can activate transcription on its own. The same basic idea is followed in LexA/B42-based yeast two-hybrid systems. Here, the two proteins LexA and B42 (acid blob) are used as DNA-binding and transcriptional activation domain, respectively. The bacterial source of these proteins reduce the risk of false positives due to endogenous yeast protein binding to one of them. For this reason, LexA-based systems are often the
method of choice. The conventional reporter gene generally used with yeast two-hybrid systems is β-galactosidase.

The Grow'n'Glow System was developed in co-operation with the Max-Planck Society as a more versatile and more accurate version of the yeast two-hybrid system, since it is using the reporter gene GFP. The practical advantages of this fluorescent protein
over β-galactosidase are substantial. In a typical β-Gal screen, individual surviving yeast colonies are picked and then tested for β-galactosidase activity either by a filter assay or growth on minimal medium plates containing X-Gal. Both procedures are often
labor-intensive, especially when hundreds of yeast colonies are obtained. In contrast, when pGNG1 is used as the reporter plasmid, the selection plates containing the yeast colonies are simply placed under a UV lamp (without the lid!) and positives become immediately identifiable by their green fluorescence. The Grow'n'Glow System is patented.

In summary, pGNG1(see chapter 4.2.1.) is a valuable addition to the family of reporter plasmids for two-hybrid systems and makes a secondary screening of yeast colonies faster and more cost-effective than conventional LacZ assays. Detection of protein-protein interactions via the green fluorescent protein provides global screening
of colonies without bias and can be adapted to most yeast-based two-hybrid strategies.

GFP
The GFP of the jellyfish A. victoria is activated in vivo by an energy transfer via the Ca2+-stimulation of the photoprotein aequorin6. The blue light generated by aequorin excites GFP and results in the emission of green light. GFP itself consists of 238 amino acids (Mr = 27 kDa) and is synthesized as an apoprotein in which post-translational formation of the chromophore occurs in an O2-dependent manner independent of any other gene products7, 5. It maximally absorbs light at 395 nm and has an emission peak of 509 nm. The nonsubstrate requirement for GFP activity makes this protein an attractive reporter for gene expression studies and this utility was initially demonstrated in both prokaryotes (Escherichia coli) and eukaryotes (Caenorhabditis elegans)7. It has subsequently been used to monitor gene expression in many organisms including mouse8, Drosophila9, zebrafish embryos10, Arabidopsis4 and yeast11. In addition to the non-invasiveness of GFP detection (long-wave UV light) the protein is very stable, non- toxic and resistant to photobleaching.
These properties make GFP a viable alternative to traditional reporter genes such as β- galactosidase (LacZ), β-glucuronidase (GUS), chloramphenicol acetyl transferase (CAT) or firefly luciferase which require substrate for their detection. Several modifications of the wild-type GFP cDNA have been engineered with optimized codon usage, improved fluorescence activity and red-shifted variants with altered excitation maxima intended for fluorescence microscopy5. The A. victoria GFP variant GFPuv is optimized for maximal fluorescence by UV-light excitation making it fluoresce 18 times brighter than wildtype GFP while retaining identical excitation and emission wavelength maxima.

Contents

The Grow'n'Glow GFP Two Hybrid System is offered as a "Complete Kit" for scientists starting to establish the two-hybrid technology, or as a "Basic Kit" for researchers already working with a LexA-based two-hybrid system.


Grow'n'Glow Two-Hybrid System "Complete Kit":

  • Vectors* Primers*
  • pGNG1 5 μg 5'-BAITprimer - 500 pmole
  • pEG202 5 μg 5'-PREYprimer - 500 pmole
  • pJG4-5 5 μg 3'-PREYprimer - 500 pmole
  • pEG202-p53 - 5 μg
  • pJG4-5-LTA - 5 μg
  • pEG202-GAL4 - 8 μg
  • Host Strain
  • Yeast strain EGY48 - 1 ml
  • Yeast strain EGY194 - 1 ml
  • Yeast strain EGY188 - 1 ml

Dane techniczne

Opakowanie zestaw

Pliki do pobrania

do góry
Sklep jest w trybie podglądu
Pokaż pełną wersję strony
Sklep internetowy Shoper.pl