The overall goal of the following experiment is to measure chaperone dependent intracellular protein, refolding after heat shock. Using a luciferase reporter first transfect cells with plasmids coating the luciferase reporter gene and the chaperone of interest. Then split the transfected cells for recovery time points in the experimental design.
On the next day, heat shock the cells to induce protein unfolding. Allow recovery to regain intracellular protein folding, and then measure luciferase reporter activity. Results measure time dependent and chaperone dependent protein refolding based on the luciferase assay.
The main advantage of this technique over existing methods, like in vitro refolding assay, is the possibility of having of a me physiological readout of the effect of compounds or proteins on the chaperone activity. The implications of this technique extend to a therapy of a wide range of diseases like neurodegenerative diseases or cancer because chaperones play an important role in the development of these pathologies. First, prewarm the culture medium, PBS and trypsin to 37 degrees Celsius in a water bath.
Wash a confluent culture of HEK 2 93 cells with five milliliters of PBS. Then apply one milliliter of trypsin containing 0.025%EDTA gently rotate the plate for even distribution of the trypsin and incubate at 37 degrees Celsius. Periodically shake the plate to verify cell detachment.
Now res suspend the cells in 10 milliliters of complete DMEM medium and transfer to a 50 milliliter falcon tube. Then enumerate cells using a Beckman counter plate. The cells at approximately 60 to 70%co fluency and incubate for six to eight hours.
Equilibrate the extreme gene, nine reagent to room temperature and the aliquot of serum free medium to 37 degrees Celsius. Pipette the serum free medium into two einor tubes. Then add the extreme gene nine reagent without touching the einor tube wall incubate for five minutes at room temperature.
In the meantime, prepare the 2D NA transfection mixes in tube one, add ran vanilla firefly and an empty vector control in tube two. Add the ran vanilla firefly and the molecular chaperone. Add the DNA to the transfection reagent mix vortex and incubate at room temperature for 20 minutes.
Continue to add this mix onto the plated cells and incubate for 24 hours. Harvest the transfected cells using trypsin seed cells at 60 to 70%co fluency in six well plates, and leave them in the incubator overnight. Aspirate the medium off transfected cells.
Then add one milliliter of heat shock buffer and incubate for 30 minutes. To stop Denovo protein synthesis, seal the lids with paraform strips and incubate in a 45 degree Celsius water bath for 30 minutes. Now remove the paraform and place the cells back into the incubator for recovery at specified time points.
Harvest the cells and wash once in PBS For an efficient cell lysis snap, freeze the cell pellet in liquid nitrogen, add 200 microliters of diluted passive lysis buffer. Now aliquot in triplicates. 30 microliters of the cell lysate per well of a 96 well plate.
In order to evaluate transfection efficiency, begin with the non shocked reference samples. To measure ran luciferase activity, prepare fresh 0.2 millimolar Lucifer in gly Gly buffer reaction buffer containing one millimolar A TP and 0.2 micromolar ezine reaction buffer. Insert the 96 well plate in the luminometer and start the program.
Wallach 1420 workstation. Introduce the pump one into the Falcon tube containing 0.2 micromolar ezine reaction buffer. Choose from the menu, the option dispenser maintenance, and tick pump one.
Select the option fill to edit the plate layout. Choose the option, explore protocol and results on the menu. Select the protocol.
Now select the wells to be read. Go back to Wallach manager and click start to measure sample emissions. At 482 nanometers, proceed to dispenser maintenance and select empty and pump one to release all the residual solution back to the tube.
Now move the tube to a falcon containing water and tick, flush. Continue by selecting empty. Then place the tube from pump one into the reaction buffer and the tube from pump two into the LUCIFERIAN solution.
Change the plate layout. Select fill for pump one and pump two. From the dispenser maintenance menu, select the protocol and start measuring the firefly luciferase reaction wavelength of 560 nanometers.
At the end of the repeating, repeat the procedure, empty, flush, empty. For pump one and pump two, view the results in the Wallach 1420 Explorer. Each experiment is associated to an assay.
Number username begin and end date export results as an Excel file. For analysis protein refolding is directly related to the time of recovery. In this experiment, cells are evaluated at different time points after heat shock.
In the absence and in presence of molecular chaperone. As expected, the percentage of refolded luciferase increases with prolonged recovery time in both controls and cells transfected with the molecular chaperone. By contrast, an invalid dataset displays poor R squared values for correlation between time and refolding.
Following this technique, other methods like in vitro TPAs assay can be performed in order to answer additional questions like inhibition of the enzymatic activity of the chaperone. After watching this video, you should have a good understanding of how to measure intracellular protein. We following after hitch shock.
