This method can answer questions in protein chemistry and drug discovery such as which conditions stabilize a protein? Which mutations impact the thermostability of an enzyme? And which ligands bind to a protein target?
The main advantages of this technique are that it's fast, easily performed in a standard laboratory, and perfectly suited to medium to high throughput applications. To prepare the protein sample transfer 10 microliters of each condition of a stability screen into the corresponding well of a 96 well plate using a multichannel pipette to save time. Then prepare one milliliter of approximately one milligram per milliliter protein solution in an appropriate buffer system.
If performing a thermal shift assay add SYPRO orange dye to the protein sample to a final concentration of 20X. Mix either by inversion or a brief vortexing. Now transfer 10 microliters of the protein solution into each well of the 96 well plate.
Seal and centrifuge the 96 well plate for two minutes at 600 times G to ensure the protein sample and screen component are mixed. Reseal the stability screen deep well block and store the screen at four degrees Celsius for up to four months. To perform the TSA experiment, open the sample drawer by firmly pressing the indent on the right hand side of the drawer.
Place the 96 well tray in the RTPCR system with well A-one to the back left. Click the new experiment button to begin setting up a TSA experiment. In the Experiment Properties tab, click the Melt Curve option when asked, what type of experiment do you want to set up?
Then click the other option when asked, which reagents do you want to use to detect the target sequence? In the Plate Setup, Define Targets and Samples tab, enter a Target Name, then set Reporter as ROX. And Quencher as None.
In the Plate Setup, Assign Targets and Samples tab, set select the dye to use as the passive reference as None. In the same tab, assign every well of the 96 well plate to the target name entered in the previous step. In the Run Method tab, delete steps until there are a total of three.
Set the first step to 25 degrees Celsius, with a ramp rate of 100%and time of five seconds. Set the second step to 95 degrees Celsius, with a ramp rate of 1%and time of one minute. Finally, set the third step to the 95 degree Celsius with a ramp rate of 100%and time of five seconds.
Choose to Collect Data using the collect data drop down menu or by clicking the data collection icon. Set the reaction volume per well to 20 microliters. Click the Start Run button to begin the TSA experiment.
NanoDSF can be used to probe protein thermal stability without the use of extrinsic dyes. Samples can be prepared in 96 well plates as described previously in the protocol but without adding any SYPRO orange dye. Open the sample drawer by pressing the Open Drawer button.
Ensure that the equipment is clean, playing particular attention to any dust near the sample rack. If the system has back scattering mirror clean it using ethanol and a lint free tissue. Load the capillaries with approximately 10 microliters from each well of the 96 well plate by touching one end of the capillary into the solution.
And then place the capillary into the corresponding capillary holders of the sample rack. Be careful not to contaminate the middle of the capillaries with fingerprints as this could interfere with fluorescence readings throughout the experiment. Immobilize the capillaries with a magnetic sealing strip.
Launch a preliminary scan to detect the position and intensity of each capillary by pressing the Start Discovery Scan button in the Discovery Scan tab. Increase or decrease the incident excitation strength from an initial power of 10%until the peak of every capillary scan is between 4, 000 and 12, 000 units. In the Melting Scan tab program a Melt Scan by setting the Temperature Slope option to 7.0 degrees per minute.
Start Temperature to 25 degrees Celsius. And End Temperature to 95 degrees Celsius. Then launch the nanoDSF experiment by pressing the Start Melting button.
Repeat these steps to prepare the samples for a full experiment. Once the full experiment is set up, navigate to the Melting Scan tab, and program a Melt Scan by setting the Temperature Slope option to 1.0 degrees Celsius per minute. Start Temperature to 25 degrees Celsius.
And End Temperature to 95 degrees Celsius. Finally, launch the nanoDSF experiment by pressing the Start Melting button. T m is used as a quantitative measure of protein thermal stability in a benchmark to compare the favorability of different conditions.
Shown here are sample results from the salt screen, exemplifying the thermally stabilizing properties of ammonium chloride towards lysozyme. Comparison of T m values of lysozyme with the pH screen reveals that the agreement between TSA and nanoDSF is generally good but nanoDSF shows a tendency to identify slightly higher T m values in slightly larger T m shifts than TSA. There's a general trend of increasing stability with decreasing pH values.
The range of T m values obtained using different buffer systems with identical pH values can be significant. For lysozyme combinations of conditions yielding the highest TSA T m values from each stability screen were tested to probe for a synergistic combined effect. There's a general increase in T m values as more components of the buffer system are added.
A noticeable synergistic affect can occur when individual components of a buffer are optimized and combined with the stability screens. Following this technique other methods such as crystallization can be used to determine the three-dimensional structure of a protein and unravel the molecular basis for ligand binding. As well we providing insight into protein stability as part of the Virus-X project, this technique is applicable to a wide range of drug discovery and development systems.
