DUALmembrane Protein Interaction Kits

Screen Full-length Integral Membrane Proteins in vivo

Discover novel interactions

DUALmembrane technology enables you to screen a membrane protein of interest for interactors expressed from a cDNA library of your choice. Full-length proteins can be used to screen for binding targets localized in the plasma membrane or the cytoplasm of the yeast. Utilizing a clever ubiqitin-based screening technology, the key interactions takes place at the cytoplasmic side of the cellular membrane, not the nucleus. Three different kit configurations allow screens for type I transmembrane proteins (starter kit SUC), type II transmembrane proteins (starter kit N), or both types I and II transmembrane proteins (starter kit STE). These systems are designed to detect either a transient or long-lasting interactions on the cytoplasmic side, the extracelluar side as well as within the membrane bilayer itself, depending upon the configuration of the prey vector.

The technology

A bait is constructed by fusing a membrane protein of interest 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.

A prey is constructed by fusing a protein of interest (either a membrane protein or a cytosolic protein) to the N-terminal half of ubiquitin carrying a I3G point mutation (NubG).

An interaction between bait and prey results in assembly of split-ubiquitin from Cub and NubG. The split-ubiquitin is recognized by ubiquitin-specific 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 which takes place at the membrane 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 yeast two-hybrid screen is a list of proteins which interact with your protein of interest (the bait). It is important to realize that standard cDNA libraries can over-represent abundant mRNAs, and this can lead to skewing screens toward those proteins which are expressed at higher levels. That is why normalized libraries are offered, so that only the most relevant interactions are selected. The normalized cDNA libraries are constructed utilizing reassociation kinetics to target and reduce over-abundant cDNAs. The net result can greatly decrease the rate of non-specific interactors and increase chances of identifying rare interactors.

The odds are stacked in your favor

What separates Dualsystems protein-interaction screening technology from home brew screens or other company kits? Each step has been optimized such that relevant interactors are identified in 98% of all screens. Traditional pitfalls have been addressed by technological advances like normalized cDNA libraries, C-terminus vs. N-terminus baits, and location-specific interactions. Dualsystems products have been used in hundreds of screens annually for the past ten years, and their vast knowledge has been applied to making state-of-the-art systems that promise the best for in vivo analysis of protein interactions. More than sixty peer-reviewed papers, over the past 8 years, refer to their products.

Assemble your kit individually or select a starter kit below!

Dualsystems provides a kit option for nearly any possible screen you can imagine. But sometimes its easiest to just order a pre-designed kit with bait and prey vectors, cDNA libraries and yeast strains already chosen for an optimized screen. For that we have nearly 100 choices of preset Starter Kits. But if your experiment requires a specialized configuration, customizing your kit is simple and speedy. Simply 1) select your bait vector below, 2) choose a normalized cDNA library or non-normalized cDNA library from our collection, then 3) the customized kit is assembled by us, and 4) you will receive your product within days! Click here to see the DUALmembrane decision tree if you're unsure which bait vector is right for you.

DUALmembrane Starter Kit Contents

DUALmembrane Starter Kits (Includes cDNA library of your choice)

ITEM #	SIZE	DESCRIPTION	PRICE	BUY
P01201 – P01244*	1 Kit	DUALmembrane starter kits N	$2,795 - $2,985**	Ordering Info
P01301 – P01344*	1 Kit	DUALmembrane starter kits SUC	$2,795 - $2,985**	Ordering Info
P01401 – P01444	1 Kit	DUALmembrane starter kits STE	$2,795 - $2,985**	Ordering Info
P01299	1 Kit	DUALmembrane starter kit N without cDNA library	$2,545 $2,290**
P01399	1 Kit	DUALmembrane starter kit SUC without cDNA library	$2,545 $2,290**
P01499	1 Kit	DUALmembrane starter kit STE without cDNA library	$2,545 $2,290**
P01501	1 Kit	DUALmembrane pairwise interaction kit	$3,105 $2,795**
P01502	1 Kit	DUALmembrane vector complementation set N (available only together with the DUALmembrane starter kit N) Contains Vectors P03231, P03232, P03233, P03235, P03236, P03237	$1,300**
P01503	1 Kit	DUALmembrane vector complementation set SUC (available only together with the DUALmembrane starter kit SUC) Contains Vectors P03230, P03231, P03233, P03235, P03236, P03237	$1,300**
P01504	1 Kit	DUALmembrane vector complementation set STE (available only together with the DUALmembrane starter kit STE) Contains Vectors P03230, P03231, P03232, P03235, P03236, P03237	$1,300**

*You must choose the correct cDNA library. Please click the item# for a list and part#s.

**Pricing shown is for academic institutions. For non-academic institutions, please inquire for additional licensing.

Starter Kit Vector Maps and Sequences

ITEM #	VECTOR	DESCRIPTION	PRICE	MAP	SEQUENCE
P03230	pBT3-N	Type II bait vector (N-terminal fusion)	$540*
P03231	pBT3-C	Type I bait vector (C-terminal fusion)	$540*
P03232	pBT3-SUC	Type I bait vector (C-terminal fusion)	$540*
P03233	pBT3-STE	Type I bait vector (C-terminal fusion)	$540*
P03234	pPR3-N	Library vector (NubG-cDNA orientation)	$540*
P03235	pPR3-C	Library vector  (cDNA orientation-NubG)	$540*
P03236	pPR3-SUC	Library vector (NubG-cDNA orientation)	$540*
P03237	pPR3-STE	Library vector (NubG-cDNA orientation)	$540*
P03243		DUALmembrane control plasmid set (3 controls)	$540*
	pOst1-NubI	Control Prey
	pTSU2-APP	Control Bait
	pNubG-Fe65	Control Prey

*Pricing shown is for academic institutions. For non-academic institutions, please inquire for additional licensing.