Capture experiments of interacting proteins
Capture assays for interacting proteins are subjected to two sequential bulk yeast plate screening processes. Saccharomyces cerevisiae contains the lexA fusion probe, the reporter gene and a cDNA expression library inserted under the control of the GAL promoter of pJG4-5.
Operation method
basic experiment
Materials and Instruments
Yeast Move 1. To transform the library, approximately 20 ml of Saccharomyces cerevisiae EGY48 culture medium containing pSH18-34 and pBait was inoculated into Glc/CM-Ura-His liquid medium, and incubated at 30°C overnight. For more product details, please visit Aladdin Scientific website.
CM medium Lithium acetate PEG TE DMSO Glycerol Ampicillin
Centrifuge Spectrophotometer Shaker Incubator
2. Dilute the overnight culture solution into 300 ml of Glc/CM-Ura-His Spare Component Liquid Medium at a concentration of about 2×106 cells/ml ( OD600≈0.10), and incubate at 30℃ until the density of bacteria is about 1×107 cells/ml ( OD600≈0.50).3. Collect yeast cells by centrifugation at low speed of 1,000~1,500 g for 5 min at room temperature. Bacteria were resuspended in 30 ml of sterile water and transferred to a conical centrifuge tube.
4. Centrifuge at 1 000~1 500 g for 5 min, discard the supernatant, and resuspend the organisms with 1.5 ml TE/0.1 mol/l lithium acetate.
5. Take 30 microcentrifuge tubes and add 1 μg of pJG4-5 plasmid inserted into the library DNA and 50 μg of high-purity uniformly sheared salmon sperm stretcher DNA respectively, the final volume should be limited to less than 10 μl, and then add 50 μl of the resuspended yeast solution respectively.6. Add 300 μl of 40% pEG 4 000/0.1 mol/l lithium acetate/TE respectively, mix upside down until completely mixed, incubate at 30°C for 30 min.7. Add DMSO to a final concentration of 10% (approximately 40 μl per tube), mix by inversion, and heat shock for 10 min at 42°C on a heating plate.
8a. 28 tubes were taken and each tube was coated with a 24 cm × 24 cm plate of Glc/CM-Ura-His-Trp Spare Component Medium and incubated at 30 °C.
8b. Remaining 2 tubes: Each tube is spread on a 24 cm x 24 cm plate of Glc/CM-Ura-His-Trp Spare Component Medium. The remaining 40 μl are diluted 1:10 with sterile water, and then the dilutions are spread on 100 mm plates of Glc/CM-Ura-His-Trp Spare Component Medium. All plates are incubated at 30°C until colonies grow (2~3 days), and at least 104 colonies are required for the saturation screening of a mammalian cell library. Transformation efficiency was determined by the plates spread after dilution.9. To harvest transformants, 30 24 cm x 24 cm plates with transformants were left to cool at 4°C for several hours to harden the agar.10. Wearing gloves and using a sterile microscope slide, gently scrape the yeast cells from the plate. Collect 30 cells scraped from the plate into 1 or 2 sterile 50 ml conical test tubes.
11. Wash the cells with 1 body Hoven's sterile TE or water, centrifuge at 1,000~1,500 g for 5 min at room temperature, discard the supernatant and repeat the washing once.12. Resuspend the precipitated yeast cells with 1 volume of glycerol solution, mix and dispense into 1 ml each and freeze at -70°C.
13. To revitalize yeast cells, make a 10-fold dilution of 1 portion of frozen transformed yeast cells with Gal/Raff/CM-Ura-His-Trp Spare Component Medium, and incubate the cells in a shaker at 30℃ for 4 h to induce the library GAL promoter.
14. The yeast cells were then diluted with a series of Gal/Raff/CM-Ura-His-Trp Nutrient-Sparing Component Medium ( 1OD600 ≈ 2 × 107 cells). The cells were spread on 100 mm plates of Gal/Raff/CM-Ura-His-Trp Nutrient Sparing Component Medium and incubated at 30°C until visible colonies developed.
15. Count the number of colonies and deduce the number of cfu per small portion of transformed yeast.16. In order to select positive clones, thaw the appropriate amount of transformed yeast, dilute according to step 13, incubate, and spread 100 mm plates of Gal/Raff/CM-Ura-His-Trp Spare Component Medium at a density of 106 cells per plate. For each initially obtained transformant, 3 to 10 cells should be guaranteed to grow after coating, and the yeast should be diluted with Gal/Raff/CM-Ura-His-Trp-Leu Spare Component Medium and incubated at 30°C for 2 to 3 days.17. When colonies have grown, carefully transfer them to a new master plate of Gal/Raff/CM-Ura-His-Trp-Leu Spare Component Medium, the earlier colonies appearing are generally more likely to be true-positive, and incubate at 30°C.18. Pick colonies from the main plate of Gal/Raff/CM-Ura-His-Trp-Leu Spare Component Medium and transfer them to a 100 mm main plate of Glc/CM-Ura-His-Trp Spare Component Medium and incubate at 30°C until colonies are visible.19. Pick bacteria from the Glc/CM-Ura-His-Trp master plate or photocopy colonies from the plate directly onto the following plates.
(1) Glc/Xgal/CM-Ura-His-Trp
(2) Gal/Raff/Xgal/CM-Ura-His-Trp
(3) Glc/CM-Ura-His-Trp-Leu
(4) Gal/Raff/CM-Ura-His-Trp-Leu20. For positive colonies (those that show blue color on Xgal-containing plates and can grow on Gal/Raff/CM-Leu plates but not on Gcl/CM-Leu plates), plasmid DNA is isolated from yeast using a rapid low-volume preparation protocol in which the total DNA from a 15 μl culture is concentrated to 5-10 μl.
21. Transform the susceptible bacterium KC8 with the prepared plasmid DNA. spread on LB/aminobenzylpenicillin plates and incubate at 37°C overnight.22. Colonies grown on LB/aminobenzylpenicillin plates were picked to or photocopied onto plates of Bacterial Extreme Medium supplemented with Ura, His, and Leu, but not Trp, to select for the pJG4.5 plasmid, and incubated overnight at 37°C.
23. Purification of library cDNA-containing plasmids using a protocol for preparing bacterial plasmids in small quantities.24. For specificity verification in separate transformation experiments, yeast already containing the following plasmids and grown on Glc/CM-Ura-His plates were transformed with the plasmids purified in step 23.
(1) EGY48 containing pSH18-34 and pBait
(2) EGY48 containing pSH18-34 and pRFHM-1
(3) EGY48 containing pSH18-34 and non-specific bait (optional)
25. Each transformation mixture was spread on Glc/CM-Ura-His-Trp Spare Component Medium plates and incubated at 30°C until colonies grew.
26. For each library plasmid to be validated, apply a master plate of Glc/CM-Ura-His-Trp Save Component Medium. That is, 5 to 6 individual colonies from each transformation plate were picked and inoculated onto. the Glc/CM-Ura-His-Trp plate and incubated at 30°C until the colonies came out on.27. Pick or photocopy colonies from the master plate onto the same series of validation plates used in the actual screening:(1) Glc/Xgal/CM-Ura-His-Trp
(2) Gal/Raff/Xgal/CM-Ura-His-Trp
(3) Glc/CM-Ura-His-Trp-Leu
(4) Gal/CM-Ura-His-Trp-Leu
28. Perform specificity verification experiments. Analyze and determine the DNA sequence of the positive plasmid.