Difference between revisions of "20.109(F19):Incubate with ligand and apply heat treatment for protein denaturation (Day2)"

Introduction

CETSA

Protocols

Part 1: Incubate cells with ligand and harvest for CETSA

1. Collect the T75 flasks that contain your MEF cell cultures prepared in the previous laboratory session.
2. Use the microscope to examine your cell cultures.
   • Make note of the confluency in your laboratory notebook!
3. Clearly label your flasks to reflect which is the experimental (ligand) and which is the control (DMSO).
4. Aspirate the media from the cells using a sterile Pasteur pipet.
5. Wash the cells by adding 2 mL PBS using a 5 mL pipet. Slightly tip the flask back and forth to rinse the cells then aspirate the PBS with a Pasteur pipet.
6. With a 2 mL pipet, add 1 mL of trypsin to the flask.
7. Tip the flasks in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 2 min.
8. Retrieve your flasks from the incubator and firmly tap the bottom to dislodge the cells.
   • Check your cells using the microscope to ensure they are dislodged. They should appear round and move freely.
   • If your cells are not detached from the flasks, incubate at 37 °C for an additional minute.
9. When your cells are dislodged, and add 3 mL of media to the cells then pipet the liquid up and down (“triturate”) to break up cells that are clumped together and suspend them in the liquid.
   • Note: do not take up or release all the liquid, in order to avoid bubbles.
10. Transfer the suspended cell cultures into separate, labeled 15 mL conical tubes.
11. Pellet the cells for 3 min at 300 g in the centrifuge.
12. Resuspend the cells in 15 mL PBS, then pellet for 3 min at 300 g in the centrifuge.
13. Collect an aliquot of PBS from the front laboratory bench and add XX protease inhibitor to a final concentration of XXYY.
14. Add 2 mL of protease containing inhibitor to each cell pellet.
15. Transfer 100 μL of the 'control' cell suspension to six labeled PCR tubes.
    • You will complete each condition in triplicate: unheated and heated.
16. Transfer 100 μL of each 'experimental' cell suspension to 3 labeled PCR tubes.
    • Again, you will complete each condition in triplicate: heated with ligand concentration #1, heated with ligand concentration #2, and heated with rapamycin.

Part 2: Apply heat treatment and snap freeze cells

Reagents list

• protease inhibitor
• ligands (from Chembridge)

Navigation links

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