Difference between revisions of "20.109(F19):Incubate with ligand and apply heat treatment for protein denaturation (Day2)"
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#Transfer 100 μL of the 'control' cell suspension to six labeled PCR tubes. | #Transfer 100 μL of the 'control' cell suspension to six labeled PCR tubes. | ||
#*You will complete each condition in triplicate: unheated and heated. | #*You will complete each condition in triplicate: unheated and heated. | ||
− | #Transfer 100 μL of | + | #Transfer 100 μL of each 'experimental' cell suspension to 3 labeled PCR tubes. |
− | #*Again, you will complete each condition in triplicate: | + | #*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=== | ===Part 2: Apply heat treatment and snap freeze cells=== |
Revision as of 22:42, 26 September 2019
Contents
Introduction
CETSA
Protocols
Part 1: Incubate cells with ligand and harvest for CETSA
- Collect the T75 flasks that contain your MEF cell cultures prepared in the previous laboratory session.
- Use the microscope to examine your cell cultures.
- Make note of the confluency in your laboratory notebook!
- Clearly label your flasks to reflect which is the experimental (ligand) and which is the control (DMSO).
- Aspirate the media from the cells using a sterile Pasteur pipet.
- 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.
- With a 2 mL pipet, add 1 mL of trypsin to the 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.
- Collect an aliquot of PBS from the front laboratory bench and add XX protease inhibitor to a final concentration of XXYY.
- Add 2 mL of protease containing inhibitor to each cell pellet.
- Transfer 100 μL of the 'control' cell suspension to six labeled PCR tubes.
- You will complete each condition in triplicate: unheated and heated.
- 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)
Next day: Begin Western blot analysis