Matchmaker™ Gold System FAQs
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1. 0 lt 0 ae 6 G L cy lt c ei cy 0 L Q Matchmaker Gold System FAQS Find out more about our latest high stringency yeast two hybrid system Principles of Yeast Two Hybrid Systems What is the two hybrid premise Yeast two hybrid technology Y2H is used primarily to discover de novo protein protein interactions Since it was invented in 1989 by Fields and Song more than 28 000 publications have cited its use to discover interactions in nearly every imaginable biological pathway In the Matchmaker Gold Yeast Two Hybrid System your target protein of interest is fused to the DNA binding domain DNA BD of the yeast GAL4 protein to create a bait protein By itself this fusion protein cannot activate transcription but is able to bind the GAL4 responsive pro moters positioned upstream of 4 reporter genes Interacting partner proteins often in the form of a library are expressed as fusions to the transcriptional activation domain AD of yeast GAL4 to create prey proteins When prey proteins interact with the bait protein GAL4 activity is restored and expression of one or more reporter proteins is activated Figure 1 1 Fields S and Song O 1989 Nature 340 6230 245 247 Why use yeast Yeast cells provide an in vivo eukaryotic system that has the handling and ease of use benefits of prokaryotic bacteria For several important reasons S cerevisiae has b2. Contact our Technical Support Department at tech clontech com for details Clontech does not however provide a library screening service Visitour website for more details clickchere Clontech O
3. are compatible for mating with yeast MATa two hybrid reporter strains Y2H Gold and AH109 used to express bait constructs Simply grow your Y2HGold bait culture overnight in SD Trp the next day mix with one vial of library and then after a further overnight incubation period to allow mating to occur spread the diploid cells on SD Leu Trp X a Gal AbA to select for colonies that harbor Y2H interactions How do I make my own library We strongly recommend using our Make Your Own Mate amp Plate Library System There is no simpler way to make a yeast two hybrid library and no easier way to screen it than the Mate amp Plate method I have a minimal amount of starting material can I still make my own library Yes Since the Make Your Own Mate and Plate Library System uses SMART technology for cDNA synthesis it is possible to start from limiting amounts of material For example you can create a library from as little as 100 ng of total RNA as the starting material How can I determine if my current library will work with Matchmaker Gold We have not tested yeast two hybrid libraries from other sources but in principle any library will work with Matchmaker Gold if the prey library vector can be selected for using a LEU2 marker Does Clontech have a custom service for yeast two hybrid libraries Yes Clontech offers a service for manu facturing custom made Mate amp Plate Libraries
4. DO double DO is so called because the medium includes every essential amino acid except for leucine and tryptophan which are omitted or dropped out from the formulation Cells harboring bait and prey plasmids are able to grow because the plasmids encode tryptophan and leucine biosynthesis genes respectively which are otherwise absent from the parental yeast strain e g Y2HGold Various other dropout supplements are also available Clontech O 0 lt 0 a 6 G L cy c 6 c ei cy 0 L Q Matchmaker Gold System FAQS onna What is Aureobasidin A and how is it used with Matchmaker Gold Matchmaker Gold Systems are unique because they use the AURI C gene as a novel reporter which confers resistance to Aureobasidin A AbA a potent antibiotic that is toxic to S cerevisiae Resistance to this highly stable antifungal depsipeptide allows straightforward Y2H library screening to be achieved without the optimization that is otherwise required when using nutritional selection alone e g HIS3 Can I use X Gal instead of X 0 Gal for blue white screening with the Matchmaker System No X Gal and X 0 Gal are not the same The former is a substrate for the E coli enzyme galactosidase lacZ while the latter is a substrate for the yeast enzyme alpha galactosidase Mell p X a Gal is used in the Matchmaker Gold System as a primary reporter for blue white colony screening sin
5. binding of a prey protein would need to occur at 3 different recognition sequences and activate all 4 reporters Matchmaker Gold is the only yeast two hybrid system with four genuine reporters controlled by three different bait recognition sequences promoters Figure 2 Why do I need to test my bait for auto activation If your bait is a eukaryotic transcription factor there is a possibility that it will activate the yeast reporters by itself in the absence of an interacting prey protein This is termed auto activation and is easily tested by checking for growth of the bait strain on selective media containing Aureobasidin A as described in the Matchmaker Gold User Manual If your bait auto activates the reporters you can still perform a two hybrid screen but you must first remove the transcriptional activation domain usually a run of acidic residues from the bait construct For example the murine p53 fragment that is used as a positive control in the Matchmaker Gold System has had the first 71 amino acids removed The N terminus of this protein would otherwise contain a transcriptional activation domain Can my bait be a transmembrane protein Yes but the two hybrid system is generally limited to investigating interactions between cytoplasmic domains of the proteins Since the Matchmaker Gold reporters are located in the cell nucleus bait prey interactions must also occur Clontech Laboratories Inc ATakara Bio Com
6. ce Mel1p is secreted from the yeast following protein protein interactions that activate the MELI gene Why do my yeast colonies sometimes turn pink ved when grown on YPD medium The red pigment exhibited by ADE2 mutants is a purine precursor which accumulates in ADE2 or ADE strains if they are grown in medium low in adenine Clontech recommends that all its yeast strains be grown in YPDA which has been supplemented with 120 mg L adenine hemisulfate to avoid accumulation of this pigment The pigment is not well characterized but some chemical analyses were published in 19674 The pigment appears to accumulate in the vacuole and fluoresces in 450 490 nm light 4 Smirnov M N et al 1967 Biochem Biophys Res Comm 27 3 299 304 5 Weisman L S et al 1987 J Cell Biol 105 4 1539 1547 Clontech Laboratories Inc ATakara Bio Company www clontech com My agar media failed to set properly what could cause this Assuming the correct amount of agar was added 20 g L this can be caused by a combination of excessive autoclaving and low pH Some sources of purified water are acidic If this is the case adjust the solution to pH 5 8 for minimal SD media and to pH 6 5 for YPDA before autoclaving for 15 min at 121 C Yeast Two Hybrid Libraries What are Mate amp Plate libraries Mate amp Plate libraries are pre aliquoted libraries that have been pretransformed into MATa haploid yeast Y187 They
7. een the favored two hybrid host organism since the inception of this now common technique Protein expression in yeast allows tertiary protein structures to form provides a neutral internal pH and produces the disulfide bonds and other post translational modifications common to other eukaryotes These features are important for maintaining native protein structures to promote authentic protein protein interactions Additionally yeast can be easily manipulated and its genome has been completely sequenced Also Clontech Laboratories Inc ATakara Bio Company www clontech com of Clontech unless noted otherwise 2009 Clontech Laboratories Inc FL912914 lt GAL4AD RNA Pol II Figure 1 Yeast two hybrid system design Library derived transcription activating prey fusion proteins that interact with the DNA binding bait fusion protein activate the expression of reporter genes Promoters Reporter Genes Aureobasidin A Resistance YUM NOW a Galactosidase VY VI Histidine Biosynthesis IIVI IVIVliIWIV0VIJVl Adenine Biosynthesis W VVIV VIII Figure 2 Four reporters give Matchmaker Gold its high stringency libraries can be made very easily and What is the difference between false directly in yeast through homologous positives and background growth recombination Mammalian two hybrid Yeast two hybrid systems that utilize only the HIS3 nutritional reporter to screen for pr
8. of amino acids salts dextrose glucose and other nutrients that enable yeast to grow rapidly YPDA is the same as YPD except for the addition of 120 mg L adenine hemisulfate which prevents yeast strains that harbor the ADE2 mutant allele from turning pink red see below In Clontech s YPDA the final concentration of adenine hemisulfate is 3X higher than the concentration used in standard protocols 40 mg L If you use media from other sources your Matchmaker yeast strains may still turn pink red What are SD media and dropout supplements Minimal media that is routinely used for culturing S cerevisiae is called synthetically defined medium or SD Clontech s SD base supplies everything that wild type yeast need to survive including carbon and nitrogen sources but lacks essential amino acids required by mutant strains Selected essential amino acids are added to SD base to create specific minimal media and pre mixed versions are available in Clontech s Yeast Media Pouches and Yeast Media Sets Alternatively you can purchase SD Base and selected amino acid dropout supplements DO separately to suit individual needs Specific minimal media are used to select for the growth of yeast that contain specific plasmids or which are expressing activated reporter genes For example SD Base mixed with the Leu Trp DO Supplement is used to select for the Matchmaker bait and prey plasmids SD Leu Trp D
9. otein protein interactions often generate a high number of i background colonies and ii false positives systems are ideal for confirmation of yeast two hybrid results but they are not very amenable to library screening due to poor ease of use and high background Background colonies are the result of leaky expression of the nutritional reporter or overly dense plating whereas false positives are caused by prey proteins that inde pendently of the bait have the ability to recognize and bind sequences upstream of the reporters Why is it important to use multiple two hybrid reporters Multiple reporters for Y2H interactions serve to minimize the emergence of false positives as explained in the following question United States Canada 1 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 O e For Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale Clontech the Clontech logo and all other trademarks are the property N C t C 0 lt 0 a 6 L cy lt 6 c ei cy 0 L Q Matchmaker Gold System FAQS onna Background colony growth is not possible with Matchmaker Gold due to the poten cy and stability of Aureobasidin A a novel yeast two hybrid antibiotic that very effectively kills all non resistant cells Additionally Matchmaker Gold reduces false positives because any independent
10. pany www clontech com in the nucleus in order for them to be detected As long as your protein folds properly and localizes to the nucleus all Matchmaker vectors add nuclear localization signals to the bait and prey proteins you should be able detect interactions with proteins in the library It is usually advisable to remove the signal peptide and the transmembrane domain from your bait There are numerous examples in the literature showing two hybrid interactions with transmembrane proteins see below e Hopkinson S B amp Jones J C 2000 Mol Biol Cell 11 1 277 286 e Traweger A et al 2002 J Biol Chem 277 12 10201 10208 e Tian X Y et al 2001 Reproduction 121 6 873 880 e Daniel J M amp Reynolds A B 1999 Mol Cell Biol 19 5 3614 3623 e Rochdi M D et al 2004 J Biol Chem 279 18 18981 18989 What minimum and maximum sizes of proteins that have been detected using yeast two hybrid analysis Proteins as small as eight amino acids and as large as 750 amino acids have been used successfully in yeast two hybrid studies 2 Heery D M et al 1997 Nature 387 6634 733 736 3 Schaefer B C Ed 1997 Gene Cloning and Analysis Current Innovations Horizon Scientific Press pp 11 28 Culturing Yeast What is the difference between YPDA and YPD medium YPD stands for Yeast Peptone Dextrose broth and is a rich medium that contains an ideal mixture
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