20.109(F19):Incubate with ligand and apply heat treatment for protein denaturation (Day2)

Introduction

At the start of the module, we discussed the DSF assay as it was used for in-vitro analysis of the putative ligands identified in the SMM screen. The DSF assay measures protein stabilization / destabilization in response to ligand by exposing the sample to a temperature gradient. In the cellular thermal shift assay (CETSA) whole cells are used to test protein stabilization / destabilization in response to increasing temperature. The use of an in-vivo test may provide information on the physiological relevance of protein:ligand binding.

Protocols

===Part 1:Calculate ligand! calculated dilutions and preparing media with ligands / controls

Part 2:Incubate cells with ligand and harvest for CETSA

1. Collect the four T75 flasks marked with your team color from the 37 °C incubator.
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 you will use for the the experimental conditions ([ligand] #1 and [ligand]#2) and which you will use for the control conditions (DMSO and rapamycin).
4. Aspirate the media from the cells using a sterile Pasteur pipet.
5. Wash the cells by adding 5 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. Add the serum-free media preparations from Part 1 to the appropriate flasks.
   • Be sure to double-check that the serum-free media preparation added matches the labels on the flasks!
7. Return your flasks to the 37 °C incubator for 1 hr.
8. Following the incubation, you can take the cell cultures from the tissue culture room to your bench and complete the remaining steps in the main laboratory.
9. Aspirate the serum-free media containing the ligand from the flasks.
10. Wash the cells by adding 5 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.
11. With a 2 mL pipet, add 1 mL of trypsin to each flask.
12. Tip the flasks in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 2 min.
13. 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.
14. 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.
15. Transfer the suspended cell cultures into separate, labeled 15 mL conical tubes.
16. Pellet the cells for 3 min at 300 g in the centrifuge.
17. Resuspend the cells in 15 mL PBS, then pellet for 3 min at 300 g in the centrifuge.
18. Obtain an 8.5 mL aliquot of PBS and add XX protease inhibitor to a final concentration of XXYY.
19. Add 2 mL of PBS containing protease inhibitor to each cell pellet.
20. Transfer 100 μL of the DMSO cell suspension to 2 labeled PCR tubes.
    • One will be the unheated control and one the heated control.
21. Transfer 100 μL of each 'experimental' and the rapamycin cell suspension to 1 labeled PCR tube.
    • All of the experimental samples and the rapamycin sample will be heated.

Part 2: Apply heat treatment and snap freeze cells

Reagents list

• protease inhibitor (from ...)
• ligands (from Chembridge)

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