The yeast two-hybrid (Y2H) system is a research method for the identification and detection of protein-protein interactions in living cells. It is now used in several research fields because of its advantages of high sensitivity, power, and wide range of applications.
Lifeasible can provide individual one-to-one validation services for your Y2H experiments. One-on-one detection of Y2H was performed by combining the two constructed plasmids separately in pairs and co-transform Y2H recipient bacteria. The interactions between the proteins to be tested are examined by detecting multiple reporter genes in the co-transformants.
| Content |
Details |
| Construction |
- Synthesis
- Subcloning
- Plasmid construction
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| Self-activation assay |
Self-activation assay of bait plasmids |
The bait recombinant plasmid PGBKT7-A and prey null PGADT7 were cotransformed into the Y2Hgold yeast strain.
- The ability to grow by spreading DDO plates indicates that the bait PGBKT7-A+ PGADT7 has been successfully transferred into the host bacteria and is not toxic to the host bacteria.
- TDO plates were spread and did not grow, indicating that the bait PGBKT7-A+ PGADT7 was not self-activated and could not activate the expression of the reporter His gene in the host bacteria.
- The QDO plate was spread and did not grow, indicating that the bait PGBKT7-A+ PGADT7 had no self-activation and did not activate the reporter ADE2 gene.
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| One-on-one validation |
Co-transformation validation |
Bait recombinant plasmid PGBKT7-A and prey recombinant PGADT7-B were cotransformed into the Y2Hgold yeast strain.
- Spread DDO plates grew, indicating that the bait PGBKT7-A+ PGADT7-B was successfully transferred into the host bacteria and was not toxic to the host bacteria.
- The growth of spread TDO plates indicated that the bait PGBKT7-A+ PGADT7-B could interact with each other and activate the expression of the reporter HIS3 gene in the host bacteria.
- The growth of spread QDO plates indicated that the bait PGBKT7-A+ PGADT7-B could interact with each other and activate the expression of both reporter genes HIS3 and ADE2.
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Experimental interpretation |
- A represents the bait and B represents the prey.
- The reporter genes involved in the screening includs HIS3, ADE2, and MEL1.
- The bait plasmid PGBKT7 carries the Trp gene, and the prey plasmid PGADT7 carries the Leu gene.
- The plates involved in the screening were DDO (SD/ -Leu/ -Trp), DDO/X (SD/ -Leu/ -Trp/ X-α-gal), TDO/X (SD/ -Leu/ -Trp/ HIS3/ X-α-gal), QDO/X (SD/ -Leu/ -Trp/ HIS3/ Ade2/ X-α-gal).
- Self-activation group: Y2H (PGBKT7-A+ PGADT7).
- Experimental group: Y2H (PGBKT7-A+ PGADT7-B).
- Positive control: Y2H (pGBKT7-53+ pGADT7-T).
- Negative control: Y2H (pGBKT7-lam+ pGADT7-T).
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Application areas
- Test the interaction between two candidate proteins
- Compare the binding between wild-type and mutant or isoform proteins
- Localize the exact interaction domain to the protein
Advantages of our technology
- High conversion efficiency and fewer false negatives.
- Rigorous control experiments are set up to exclude false positives and negatives results.
- The Y2H system uses multiple reporter genes with different upstream regulatory regions for each reporter gene, which significantly reduces false positives.
- The reporter genes are integrated into the chromosome, which makes the gene expression level stable and eliminates the false positives caused by the fluctuation of gene expression level due to the change of plasmid copy number.
- Strictly set the point species to verify the growth state of the experimental organism and further verify whether the mutualism and the strength of the mutualism.
Lifeasible provides Y2H one-on-one validation service for our customers, please feel free to
contact our staff to submit your validation project.
