Experiments to identify chromatin-bound proteins from total cell extracts by immunoprecipitation methods
Experiments to identify chromatin-bound proteins from total cell extracts by immunoprecipitation methods
This experiment is both a simple and very sensitive method for determining whether a known protein in Saccharomyces cerevisiae binds to a known DNA sequence. Source: Compact Molecular Biology Laboratory Guide, Fifth Edition
Operation method
Experiments to identify chromatin-bound proteins from total cell extracts by immunoprecipitation methods
Principle
Intact cells are treated with formaldehyde to immobilize the structure of chromatin in vivo by protein-DNA and protein-protein cross-linking. Total cell extracts are drawn and ultrasonically broken to solubilize the chromatin and shear the DNA into fragments of appropriate length. The soluble chromatin is incubated with a primary antibody to the protein of interest, and the protein-antibody complex can be precipitated by co-incubation with the G-protein coupled to the Sepharose, so that the DNA molecules crosslinked to the specific protein of interest are coprecipitated at the same time. The protein-DNA cross-links are broken and the DNA purified by elution of the antibody complex beads, and the precipitated DNA can be used to identify the enriched DNA sequences by PCR amplification using specific primers.
Materials and Instruments
Yeast Culture RNase A (no DNase) Move 1. For each sample, grow 50 ml of yeast cells to an OD600 of 1.0-1.5. Prepare a 50 ml capped tube with 1.4 ml of 37% formaldehyde solution at room temperature. 2. Add yeast culture to below the 50 ml mark, leaving plenty of room for the 2.5 ml of glycine to be added. 2. Fill the tube with yeast culture solution below the 50 ml mark, leaving enough room for the 2.5 ml of glycine to be added. Screw the cap on tightly and leave at room temperature for 15 min, inverting the tube occasionally to mix the solution. 3. 3. Add 2.5 ml of 2.5 mol/L glycine (to a final concentration of 125 mmol/L) to the tube to stop cross-linking; mix and leave at room temperature for 5 min. 4. 4. Centrifuge the cells at 1500 g for 5 min at 4°C and discard the supernatant. Place cells on ice and resuspend in 20 ml ice-cold TBS. Repeat this step. 5. Centrifuge cells at 1500 g for 5 min at 4°C and discard supernatant. Resuspend cells with 1 ml ice-cold TBS and transfer to an ice-cold 2 ml bottomed microcentrifuge tube on ice. 6. 6. Collect the cells by brief centrifugation at maximum speed and discard the supernatant. 7. 7. If cells are frozen, thaw on ice. Add 250 ul of ice-cold lysis buffer containing protease inhibitors to the cell sediment. Add 1 volume of glass beads (approx. 350 ul). 8. Vortex the cells in a multi-well vortex mixer at 4°C for 30 min at maximum speed to lyse the cells. 9. 9. Add 250 ul of ice-cold Lysis Buffer with Protease Inhibitor and vortex briefly. Place on ice. 10. Quickly wipe ice and water off the outside of the 2 ml microcentrifuge tube with blotting paper. Invert the tube and pierce two holes in the bottom of the tube with a hot needle (26-G). Immediately place the tube into a 14 ml round-bottomed tube on ice so that the microcentrifuge tube hangs just above the mouth of the larger tube. 11. Centrifuge the sample at 1000 g for 3 min at 4°C using a tumbling bucket rotor and place the sample on ice. After making sure that all of the sample except the glass beads is in the 14 ml tube, discard the 2 ml microcentrifuge tube containing the glass beads. 12. 12. Resuspend the precipitate by aspirating the solution layer several times with a 1 ml microfuge. The suspension is then transferred to a 1.5 ml microcentrifuge tube on ice. 13. Ultrasonically fragment the cut chromatin to an average size of 500 bp: When using an ultrasonic fragmenter, start 3 times at 12 s (e.g., Branson Sonifier250, energy setting 2.0, 100% action cycle). Leave the sample on ice for at least 2 min between each start-up. 14. Centrifuge at maximum speed for 5 min at 4°C to precipitate unbroken cells and cellular debris. Transfer the supernatant to a new 1.5 ml microcentrifuge tube on ice. 15. 15. Centrifuge at maximum speed for 15 min at 4°C and transfer the supernatant to a new 1.5 ml microcentrifuge tube on ice. 16. 16. Add the protein primary antibody of interest to the chromatin extract and mix well. incubate at 4°C for 1 h until overnight. 17. Using a wide-mouth pipette tip (e.g., a 200 ul pipette tip with the tip cut off), add 30 ul of Protein G-Sepharose beads (50% suspension in PBS) per sample and mix well. rotate and mix at 4 °C for 1 h. 18. 18. Centrifuge at 1000 g for 1 min at room temperature and remove 1/10 volume (50 ul) of supernatant into a new 1.5 ml microcentrifuge tube. Add 200 ul TE/1% SDS and mix well at 4°C until step 27. 19. Discard the remaining supernatant. 19. Discard the remaining supernatant. Wash the beads by adding 1 ml of Lysis Buffer and incubating for 5 min at room temperature on a rotary mixer. Centrifuge at 1000 g for 1 min at room temperature to precipitate the beads. 20. 20. Repeat step 19 with 1 ml of Lysis Buffer. 21. 21. Repeat step 19 with 1 ml of Lysis Buffer 500. 22. 22. Repeat step 19 with 1 ml LiCl/Descaler solution. 23. 23. Add 1 ml TE Buffer and repeat step 19. After centrifugation, remove as much of the wash solution as possible without disturbing the beads. 24. Add 100 ul Elution Buffer and repeat step 19. 24. Add 100 ul of Elution Buffer and mix well. 65°C water bath or heater for 10 min to elute the precipitate from the antibody gel beads. 25. Briefly centrifuge the beads at maximum speed and transfer the eluate (100 ul) to a new 1.5 ml microcentrifuge tube. 26. 26. Add 150 ul TE/0.67% SDS to the tube of gelatin beads, invert the tube several times and centrifuge briefly at maximum speed. Combine the second eluate (150 ul) with the first (precipitate). 27. Incubate the precipitate and the total mixture (step 18) in an air incubator or oven at 65 °C for at least 6 h. 28. 28. Add 250 ul of Proteinase K solution and incubate at 37°C for 30 min for the precipitate and at 37°C for 2 h for the total mixture. 29. Add 55 ul 4 mol/L LiCl and 500 ul 25:24:1 phenol/chloroform/isoamyl alcohol, vortex vigorously for 1 min (a multi-well vortex mixer will be convenient), and centrifuge at room temperature at maximum speed to separate the layers (5 min for the precipitate; 10 min for the total mixture). The total mixture usually requires a second phenol/chloroform/isoamyl alcohol extraction. 30. 30. Transfer the upper aqueous phase to a new 1.5 ml microcentrifuge tube and add 1 ml of 100% ethanol. Mix well and centrifuge at room temperature at maximum speed for 15 min to precipitate the DNA. The precipitate obtained after centrifugation in the precipitation tube is so small that it is barely noticeable, while the total mixture contains a large amount of white precipitate. 31. 31. The precipitate is washed with 750 ul of 75% ethanol and centrifuged at maximum speed for >5 min at room temperature and the supernatant is discarded. The sample was dried in air for about 10 min. 32. 32. Resuspend the precipitate from the precipitation tube with 100 ul TE. Store at -20°C. 33. 33. Resuspend the precipitate from the total mixture tube with 50 ul of TE containing 0.2 mg/ml RNase A (diluted with 20 mg/ml reservoir solution) and incubate at 37°C for 30 min. Add 950 ul of TE and store at -20°C. 34. 34. Set up a 50 ul PCR reaction with a primer concentration of 1 pmol/ul, using either 2 ul of precipitate or 1 ul of total mixture as template. 35. 35. Perform hot-start PCR using the following thermal cycling parameters. 36: 36. The PCR products are added to the appropriate sample buffer and analyzed by ethidium bromide staining using non-denaturing polyacrylamide gel electrophoresis or agarose gel electrophoresis. 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Formaldehyde Glycine (heat sterilized) TBS solution Lysis buffer LiCl Descaler Detergent TE buffer Elution buffer Protease K solution LiCl Phenol Chloroform Isoamyl alcohol Ethanol
Spin-cap tubes Flat-bottomed microcentrifuge tubes Multi-well bane spin mixer 26-G needle Round-bottomed tubes (17 mm x 100 mm) Microcentrifuge tubes Ultrasonograph (probe-type) Spin mixer Water baths and heaters Incubators or ovens