Traditional construction of a GAL4-AD fusion protein library for yeast two-hybrid (Y2H) assays entails a lengthy workflow (cDNA synthesis, cloning the cDNA library into a yeast expression vector, library-scale amplification in E. coli, and library-scale transformation into yeast), requiring significant time and effort before screening can even begin. Our Matchmaker systems, including our latest Matchmaker Gold Yeast Two-Hybrid System, are highly optimized tools for screening cDNA libraries in yeast and provide a simple workflow to identify novel DNA-binding proteins or protein-protein interactions (Figure 1).
- Capturem technology
- Antibody purification and immunoprecipitation
- His-tag purification
- Other tag purification
- Phosphoprotein and glycoprotein purification
- Mass spectrometry digestion reagents
- Matchmaker Gold yeast two-hybrid systems
- Expression systems
Mate and Plate yeast two-hybrid cDNA libraries
- No library-scale plasmid amplification or transformation required for premade Mate & Plate libraries
- Select from a wide variety of cDNA libraries: human, mouse, normalized, and universal
- Simply and efficiently make your own pretransformed library with SMART technology
- Perfect for use with the Matchmaker Gold Yeast Two-Hybrid System
Simple screening with Mate & Plate libraries
We offer an extensive variety of validated, pretransformed cDNA libraries in yeast. Our Mate & Plate libraries are cDNA libraries of GAL4-AD prey fusion proteins that are ready for immediate screening in Y2H systems. These libraries have been transformed into the MATα haploid yeast strain, Y187, which can be easily mated to a haploid MATa reporter strain (such as Y2HGold or AH109). We also offer the option to generate your own custom libraries with our Make Your Own "Mate & Plate" Library System.
Our Mate & Plate protocol makes library screening an easy task: simply combine the Mate & Plate library culture with a culture of your bait-expressing reporter strain (Figure 1). Coculturing the two strains overnight produces an array of diploid yeast clones, each coexpressing your bait with a different library prey protein. The clone pool can then be plated on selective media to screen for individual clones that express the appropriate reporter genes and markers, indicating the presence of interacting hybrid protein pairs.
We also offer a selection of normalized Mate & Plate libraries which further simplify the search for novel PPIs. Duplex-specific nuclease (DSN) normalization selectively removes abundant, and therefore redundant, cDNAs from the total pool and enriches the library for less abundant sequences (Figure 2). This process eliminates a major source of potential false positives. Library complexity effectively increases, reducing the number of independent clones that must be screened in order to detect genuine positive interactions. This lowers the frequency of false positives that emerge from primary, low-stringency screens. By using normalized Mate & Plate libraries together with the stringent screening methodology provided by the Matchmaker Gold Yeast Two-Hybrid System, your primary screens will greatly favor the identification of genuine positives, produce few false positives, and yield virtually no background colonies.
To illustrate how DSN normalization results in more balanced gene representation, cDNA samples before and after normalization were compared on a NimbleGen microarray containing 47,633 human genes (Figure 2). In this analysis, cDNA species from the most highly expressed genes were preferentially eliminated, while less abundant cDNAs remained largely unaffected. Consequently, the representation of low-copy-number sequences increased within the total cDNA pool. Genes whose cDNA levels were sharply reduced following normalization included two highly expressed housekeeping genes, ß-actin and GAPDH (Figure 3).
Universal coverage, efficient Y2H screens
Our universal libraries provide the broadest and most complete coverage of genes expressed in almost any tissue. To create these all-purpose, normalized libraries, we combined RNAs from a diverse collection of either mouse or human whole tissues that are specifically chosen to represent an expansive range of expressed genes. These same RNA pools are used for our qPCR Human Reference cDNA and qPCR Human Reference Total RNA, as well as our Mouse Universal Reference Total RNA. Following cDNA synthesis and amplification using SMART technology, we normalize each cDNA pool before constructing and transforming the library into yeast. Combining "across-the-board" gene representation with the enrichment of low-copy-number cDNA, our universal normalized libraries offer the greatest capacity for identifying genuine binding partners of your protein of interest.
To demonstrate this, we searched for binding partners of a murine p53-bait using our Mate & Plate Library - HeLa S3 (Normalized). A medium-stringency screen of only 279,000 clones yielded 62 colonies that could possibly contain p53-binding proteins (Table I). In contrast, we recommend screening 1–2 million clones for a standard library. Of eight colonies that were selected for further analysis, four contained three different, well-known binding partners of p53: PCNA, PRMT3, and PTEN. Thus, it is possible to screen a significantly smaller number of clones in a normalized library and still generate valuable data.
|Number of clones screened||279,000|
|Total number of colonies on medium-stringency selection plates||62|
|Number of colonies analyzed||8|
|Verified p53 binding partners||
|Unverified p53 binding partners (potential false positive interactors)||
Table I. Identification of p53 binding partners using our Mate & Plate Library - HeLa S3 (Normalized). Binding partners of a murine p53 bait were initiated using the normalized human HeLa S3 library. Binding partners for p53 were selected for by plating on medium-stringency (SD/–leu/–trp/–his) plates. Colonies were analyzed by performing yeast colony PCR on prey inserts from eight colonies and sequencing PCR products.
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