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

From Course Wiki
Jump to: navigation, search
20.109(F19): Laboratory Fundamentals of Biological Engineering

Fa19 20109 Banner image.png

Fall 2019 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Testing chemical probes              


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. More specifically, the CETSA asks if a ligand is able to bind in a complex system (whole cell) whereas the DSF assay probes binding in an artificial system (reaction tube).

Protocols

Part 1:Prepare for CETSA treatments

Before you start your experiment in the tissue culture room, you need to calculate the dilutions for your treatment conditions. When your calculations are completed, alert the teaching faculty.

Use the following information (and the table below) to calculate the volume of each component should be added for each condition:

  • Rapamycin stock concentration = 1 mM
  • Ligand stock concentration = 1 mM and 10mM
    • You will use the 1 mM stock to prepare the 3 μM experimental treatment and the 10 mM stock to prepare the 30 μM treatment.
  • To account for possible effects of DMSO (both rapamycin and the ligand were dissolved in DMSO), the volume of DMSO added to the control should be equivalent to the volume of rapamycin / ligand added for the other treatments.
  • When you complete your calculations, alert the teaching faculty. You may need to wait for a available biosafety cabinet...please be patient.
Treatment DMSO rapamycin (treatment concentration = 3 μM) ligand (treatment concentration = 3 μM) ligand (treatment concentration = 30 μM)
DMSO negative control
rapamycin positive control
ligand, 3 μM experimental
ligand, 30 μM experimental

Part 2:Incubate cells with ligand and harvest for CETSA

  1. Obtain an aliquot of serum-free media and four 15 mL conical tubes.
  2. Add 10 mL of serum-free media to each conical tube and label accordingly:
    • DMSO control
    • rapamycin control
    • 3 μM ligand experimental
    • 30 μM ligand experimental
  3. Add the appropriate volume (calculated above) of each component to the correct conical tube. Be sure to double-check with your labels!
  4. Collect the four T75 flasks marked with your team color from the 37 °C incubator.
  5. Use the microscope to examine your cell cultures.
    • Make note of the confluency in your laboratory notebook!
  6. Clearly label your flasks to reflect which you will use for the the experimental conditions (3 μM ligand and 30 μM ligand) and which you will use for the control conditions (DMSO and rapamycin).
  7. Aspirate the media from the cells using a sterile Pasteur pipet.
  8. 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.
  9. Add the correct serum-free media preparations to each of the flasks.
    • Be sure to double-check that the serum-free media preparation added matches the label on the flask!
  10. Return your flasks to the 37 °C incubator for 1 hr.
  11. 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.
  12. Aspirate the serum-free media containing the ligand from the flasks.
  13. 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.
  14. With a 2 mL pipet, add 1 mL of trypsin to each flask.
  15. Tip the flasks in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 2 min.
  16. 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.
  17. 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.
  18. Transfer the suspended cell cultures into separate, labeled 15 mL conical tubes.
  19. Pellet the cells for 3 min at 300 g in the centrifuge.
  20. Resuspend the cells in 3 mL PBS, then pellet for 3 min at 300 g in the centrifuge.
  21. Obtain an 1.5 mL aliquot of PBS containing protease inhibitor.
  22. Resuspend each cell pellet in 250 μL of PBS containing protease inhibitor.
  23. Transfer 100 μL of the DMSO-treated cell suspension to 2 labeled PCR tubes.
    • One will be the unheated control and one be heated.
  24. Transfer 100 μL of each 'experimental' and the rapamycin-treated cell suspension to 1 labeled PCR tube.
    • Both of the experimental samples and the rapamycin sample will be heated.

Part 3: Complete heat-induced denaturation and snap freeze cells

  1. The samples will be heated using the thermocyler. Take your tubes to the front laboratory bench and when all groups are ready, the samples will be heated for 3 minutes at 59 °C then cooled for 3 minutes at 25 °C.
  2. The teaching faculty will assist you as you complete the following steps.
    • To snap freeze your cells, carefully drop your tubes in the liquid nitrogen for ~10 seconds.
    • Carefully remove the tubes and place them in the thermocycler to thaw for 3 minutes at 25 °C.
    • Complete the snap freeze procedure a total of three times; however after the third snap freeze, place the tubes in the -80 °C freezer instead of performing a final thaw.

Reagents list

  • protease inhibitor cocktail (Sigma)
  • small molecule ligands (Chembridge)

Navigation links

Next day: Begin Western blot analysis

Previous day: Prepare for cellular thermal shift assay