Our research focuses on protein targets, prediction, and validation of small molecular compounds. The experiment used here shows a method of molecular docking combined with similar thermo shift assay to predict and validate the interaction between small moleculars and protein targets. The steps of the TSA are complicated and any steps errors during the operation can have a great impact on the results.
Our protocol combines prediction with verification, effectively reducing both time consumption and economic costs. Furthermore, the experimental instruments and materials are easy to option. Finally, the assayed TSA samples are easy to prepare and test, which ensures the reproducibility of the samples.
To begin, download the crystal structures of xanthatin and KEAP1. After processing the structures, navigate to Tasks and select the Ligand Docking option. Choose the Receptor Grid As from the file and click on Browse.
Then click Desktop. Open the Molecular docking folder, and double click on the glide-grid 2FLU file. Select the glide-grid 2FLU.
zip file and click Open. Now click on Use Ligands from Files. Then click Browse and select Desktop.
Select the Molecular docking folder and open the ligprep_1 file. After that, select the ligprep_1-out. maegz file and click Open.
Access Settings and choose the precision as XP option. Modify the job name to glide. XP_2FLU, and then click Run.
To view docking results, click on File and select Import Structures options. Navigate to the Desktop and open the Molecular docking folder. Afterward, select the glide-doc_XP_2FLU file.
Choose the glide-doc_XP1_pv. maegz file, and click open. Then double-click on the dot in the workspace to select the ligand.
Choose sitemap_1_protein. Click on table and slide to the far right to view the docking score. To visualize the 2D results, select sitemap_1_protein as demonstrated earlier.
Click on ligand Interaction. Select View and check the LID Legend box. Then click File.
Choose Save Screenshot and input 6, 000 for width. Uncheck the transparent background box and click Okay. Save the file on the desktop as 2D-xanthatin-2FLU.
Molecular docking analysis predicted the interaction between xanthatin and KEAP1 protein. To begin, add raw 264.7 cells into a 100 millimeter cultured dish and incubate with 6 milliliters of DM-EM at 37 degrees Celsius with 95%humidity and 5%carbon dioxide. Once the cells attain 80%confluency aspirate the old medium.
Then wash the cells two times with 2 milliliters of PBS warmed to room temperature. Next, add 2 milliliters of PBS to each dish containing cells, and mix them. Collect the cells into two 1.5 milliliter microcentrifuge tubes, then centrifuge at 377G for three minutes at room temperature.
After discarding the supernatant, resuspend the cells in 2 milliliters of PBS. Now freeze microcentrifuge tubes in liquid nitrogen until white solid forms, and immediately thaw in a 37 degrees Celsius water bath. Centrifuge the tubes at 12, 000 G for 10 minutes at 4 degrees Celsius.
Next, take two microcentrifuge tubes, one with 100 micromolar xanthatin, and one with an equal volume of dimethyl sulfoxide. Add 450 microliters of the centrifuged supernatant to each tube and incubate at 37 degrees Celsius for 30 minutes. Then transfer 60 microliters of supernatant from each microcentrifuge tube to 14 PCR tubes.
Heat the tubes simultaneously at varying temperatures for three minutes, with two PCR tubes at each temperature. Then place the tubes in the centrifuge. Now take 48 microliters of supernatant from each tube and add it to a new 1.5 milliliter microcentrifuge tube.
Add 12 microliters of SDS-PAGE protein loading buffer to each tube. Heat the vortexed samples in a water bath at 100 degrees Celsius for five minutes. To begin, prepare the SDS-PAGE gel.
Spin the thawed marker and the prepared cellular thermal shift assay samples. Add 2.5 microliters of marker to the first well and 20 microliters of samples to each of the remaining wells. Then run the gel at 80 volts.
Next add 850 milliliters of ultrapure water, 100 milliliters of anhydrous ethanol, and 50 milliliters of rapid transfer buffer to a beaker and stir well. Cut the polyvinylidene fluoride, or PVDF membrane, according to the size of the gel and mark it. Then soak it in methanol for 30 seconds to activate it.
Place the transfer clamps, sponge pad, and three layers of transfer filter paper in a tray containing the transfer buffer. Once the electrophoresis is complete, pour out the electrophoresis solution from the electrophoresis device and remove the plate. Gently remove the gel with a plastic knife and cut it to clearly visualize the protein marker and the target proteins.
After washing the gel in the transmembrane solution, lay it flat on filter paper. Dip the activated PVDF membrane into the transfer solution. Hold the labeled side with tweezers and cover the gel face down.
Then place the assembled transfer setup into the transfer tank. Add the remaining transmembrane solution to the transmembrane cassette. Adjust the current and time, and press run to start the transfer at room temperature.
Now add six milliliters of 5%BSA solution with TBST to the incubation box. Once the transfer is complete, remove the PVDF membrane from the setup while clamping the marker side and place it in the incubation box. After removing the BSA solution from the incubator box, add 6 milliliters of primary antibody.
Place the incubation box in a foam box with ice packs overnight while shaking. The next day, collect the primary antibody in a tube and wash the membrane with 6 milliliters of TBST solution. Afterward, add 6 milliliters of secondary antibody to the incubation box.
At the end of the incubation, collect the secondary antibody and wash the membrane five times with TBST, as shown earlier. Mix equal volumes of chemiluminescent reagents A and B.Open the gel imaging system. Click on Samples, check the calibration, and wait for calibration to complete.
Then click on Marker and check the calibration. Now pick up the marker side of the strip with tweezers and fully immerse in the chemiluminescent reagent solution. Clamp one side of the marker to place the strip face down in the imager and close the lid of the imager.
Set the exposure time to one second and click Capture. Click Save. Name the image and save it as a TIFF file.
The cellular thermal shift assay showed that xanthatin remarkably shifts the thermal stability of KEAP1 protein within the temperature range of 48 to 57 degrees Celsius, as compared to the dimethyl sulfoxide group.
