DUALhunter starter kits

Discovery of protein interactions using nuclear proteins, transcription factors and self-activating proteins

Article number: P01600 Size: 1 kit Storage temperature: -20°C / 4°C / RT Shipping temperature: RT

Applications

Discovery of novel protein interactions by screening of cDNA libraries
Compatible with nuclear proteins, transcription factors, acidic proteins and self-activating proteins
Analysis of direct protein interactions
Domain mapping studies

Benefits

Alternative for screening proteins which are self-activating in classical yeast two-hybrid systems
Screening of acidic proteins and transcription factors

Available cDNA libraries of your choice*

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DUALhunter technology enables you to screen proteins which are unsuitable for use in classical two-hybrid systems, such as transcription factors, nuclear proteins or acidic proteins. The bait protein is attached to the membrane via a small transmembrane domain and is screened outside of the nucleus for interaction with other proteins.

Mechanism of the DUALhunter system

The bait

DUALhunter technology - The bait A bait is constructed by fusing a protein of interest at its N-terminus to a small membrane anchor (Ost4p) and at its C-terminus to the C-terminal half of ubiquitin (Cub) and a transcription factor. The bait is inserted into the membranes of yeast. Since the transcription factor is fused to the membrane protein, it is immobilized at the membrane and cannot reach the nucleus. Consequently, the reporter genes integrated into the yeast genome remain silent.

The prey

DUALhunter technology - The prey A prey is constructed by fusing a second protein of interest to the N-terminal half of ubiquitin carrying a I3G point mutation (NubG).

Interaction between bait and prey

DUALhunter technology - Interaction between bait and prey An interaction between bait and prey results in assembly of split-ubiquitin from Cub and NubG. The split-ubiquitin is recognized by ubiquitinspecific proteases present in yeast, resulting in proteolytic cleavage of the transcription factor from the bait. The liberated transcription factor moves to the nucleus and activates the reporter genes in the yeast genome. The protein interaction is thus converted into reporter gene activity. Commonly used reporter genes are either auxotrophic markers which allow selection on a minimal medium or color reporters like lacZ.

Screening of cDNA libraries to identify novel interactions

The defined prey can be replaced by a collection of different preys (i.e. different proteins fused to the activation domain) expressed from a cDNA library. Each yeast cell expresses the same bait and a particular prey from the cDNA library. If bait and prey interact, the yeast cell multiplies and gives rise to a visible colony on minimal medium. All yeast cells which do not express an interacting bait/prey pair fail to grow and are discarded. Finally, yeast colonies are picked and the prey plasmid is isolated and sequenced, yielding the information on the protein which interacts with the bait. The final result of a DUALhunter screen is a list of proteins that interact with your protein of interest (the bait).

Content	Size
pDHB1 bait vector	5 µg
pPR3-N prey vector	5 µg
pOST1-NubI control prey vector	5 µg
pTSU2-APP control bait vector	5 µg
pNubG-Fe65 control prey vector	5 µg
NMY51 yeast reporter strain	stab culture
HTX beta-galactosidase assay kit	1 kit
DS Yeast transformation kit	1 kit
cDNA library of your choice	60 µg