20.109(F19):Incubate with ligand and apply heat treatment for protein denaturation (Day2)
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Contents
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!
Part 2:Incubate cells with ligand and harvest for CETSA
- Collect the four T75 flasks marked with your team color from the 37 °C incubator.
- Use the microscope to examine your cell cultures.
- Make note of the confluency in your laboratory notebook!
- 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).
- Aspirate the media from the cells using a sterile Pasteur pipet.
- 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.
ligand treatment
- At this point, you can take the cell pellets from the tissue culture room to your bench and complete the remaining steps in the main laboratory.
- Aspirate the media containing the ligand from the flasks.
- 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.
- With a 2 mL pipet, add 1 mL of trypsin to each flask.
- Tip the flasks in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 2 min.
- 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.
- 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.
- Transfer the suspended cell cultures into separate, labeled 15 mL conical tubes.
- Pellet the cells for 3 min at 300 g in the centrifuge.
- Resuspend the cells in 15 mL PBS, then pellet for 3 min at 300 g in the centrifuge.
- Obtain an 8.5 mL aliquot of PBS and add XX protease inhibitor to a final concentration of XXYY.
- Add 2 mL of PBS containing protease inhibitor to each cell pellet.
- Transfer 100 μL of the DMSO cell suspension to 2 labeled PCR tubes.
- One will be the unheated control and one the heated control.
- 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
- ligands (from Chembridge)
Next day: Begin Western blot analysis