The overall goal of this procedure is to determine the binding affinity of a protein ligand interaction without purifying the protein from a cell lysate. This is accomplished by first expressing the GFP fused protein of interest in any adherent cell line. Following preparation of cell lysate and ligand dilutions cell lysate ligand mixtures are prepared and loaded into capillaries.
Next, the thermo resis of the GFP fused protein in the presence of varying ligand concentrations is measured. The final step is data analysis of microscale thermo pheresis or MST measurements. Ultimately, microscale thermo resis is used to determine the binding affinities of interactions between GFP fused proteins and various ligands.
The main advantage of this technique of existing methods like titration, calorimetry, or surface plasma mono is ance, is that it avoids protein purification, the significantly simplifying and accelerating quantitative characterization of binary interactions. This method presents significant advantages when working with proteins that are difficult to express and purify, such as membrane proteins and transcription factors. One of the biggest advantages of microscale thermo pheresis is that it works in a variety of buffers and tolerates the presence of detergents and my cells in the system Demonstrating the technique will be Karen Stefka, a biologist from my laboratory To begin wash the cells briefly with ice cold phosphate buffered saline or PBS using 10 milliliters of buffer per T 75 flask.
Keep the cells on ice for five minutes or until they start detaching from the flask. Scrape the cells with the cell scraper to detach if necessary. Re suspend the cells in 10 milliliters of ice cold PBS and transfer to a pre chilled 14 milliliter round bottom centrifuge tube.
Pellet the cells by centrifugation at 400 to 600 Gs and four degrees Celsius for five minutes. Remove the supernatant and resuspend the pellet in 200 microliters of ice cold lysis buffer. Transfer the suspension to a pre chilled 1.5 milliliter einor tube for extraction of cytosolic proteins.
Place the cells on ice to minimize local overheating and ly cells with three times ten second pulses of sonication at 30%amplitude. Using a two to three millimeter pre chill tip, keep the tip below the surface to minimize frothing. Omit this step when using detergent containing buffer and incubate on ice for 30 minutes.
Instead, correct the lysate solution to contain a physiological salt concentration of 100 millimolar sodium chloride if necessary from a stock solution of five molar sodium chloride. Finally, collect the lysates by centrifugation at about 25, 000 Gs and four degrees Celsius for 10 minutes. Select the light emitting diode or LED excitation source with the wavelength equal to 470 nanometers on the MST instrument.
Load capillaries with cell extract diluted two and 10 times with MST buffer and place them in the MST instrument. Perform the find capillaries operation on the control software of the MST instrument. The optimal fluorescence range in diluted lysate is from 400 to 1500 fluorescence units.
Proceed to determine the optimal ligand concentration range as described in the text protocol. Place a tube rack with the necessary number of 0.5 milliliter low bind centrifuge tubes on ice pipette 25 microliters of MST buffer to the bottom of each tube at 25 microliters of the ligand stock solution to the first tube and perform serial twofold dilution of the ligand using the rest of the tubes. Keep the rack with ligand samples on ice.
Calculate the cell lysate dilution to yield the optimal level of the fluorescent target protein in the binding reactions. Final protein concentration should be close to the expected binding dissociation constant or lower, and should be adjusted to obtain the necessary number of fluorescence counts. In the final solution, proceed to dilute the cell lysate with MST Buffer.
Place 0.5 milliliter low bind tubes in the tube rack across from tubes with ligand serial dilution samples. Carefully add 15 microliters of the cell lysate to the bottom of each tube. Try not to touch tube walls to avoid sample loss.
Add 15 microliters of the ligand sample with the highest concentration to the corresponding tube. Number one, with the cell lysate. Mix well and change the pipette tip.
Repeat this step with the rest of the tubes except for the last one, which should contain no ligand. Add 15 microliters of MST buffer to the last tube and mix. Well fill approximately two thirds of the first capillary with the binding mixture from tube number one.
Tilt it to move the solution towards the center and place the capillary on the capillary tray to the position. Number one, repeat this step with the rest of the capillaries. Capillary ends can be plugged with wax for longer experiments.
Place the tray inside the MST instrument and close the instrument door. Next, select the LED excitation source with wavelength equal to 470 nanometers. On the MST instrument, perform the find capillaries command to let the instrument find the exact positions of the capillaries and measure fluorescence of the samples based on the fluorescent signal intensity.
Adjust the LED power to bring it into the 400 to 1500 units interval. Click the start button to perform the Thermo Resis Experiment. More than one infrared laser power can be chosen for the experiment.
In order to find the optimal temperature gradient for the particular system, it takes 10 to 12 minutes to run one set of 16 capillaries. Collect data from two to three runs for the same set of capillaries to ensure the reproducibility of the measurements. To begin data analysis, open the analysis software and load the project folder.
In the appeared information run viewer select collected at a specific IR laser power thermo retic curves. There was an option of opening all thermo retic traces collected under various conditions at once, and then choosing any curves for analysis by switching them on and off. In the evaluation points graph window, select the thermo resis or thermo pheresis with T jump blue and red lines.
Define two regions of the experimental curves that are selected manually for the analysis by the software. Ensure that blue and red lines are positioned correctly to provide for the maximal change in Thermo Resis. To obtain the averaged points with standard deviations, select use average or distinguish runs for separate runs.
To plot a dissociation constant fit, select use average, enter and fix the labeled molecule concentration value and fit the curve. The binding dissociation constant or KD value with its standard deviation appears in a separate information popup window. Save the average fit data in a text file and transfer to Excel.Heck.
2 9 3 cells expressing STAT 3G FP were used as a source of fluorescently labeled stat three. For A DNA binding assay binding of highly charged oligonucleotides resulted in significant changes in STAT three mobility. In the temperature gradient.
Thermo retic signal is plotted as a function of oligonucleotide concentration. Each data point represents the mean of three measurements. Nano temporal analysis software was used to fit the and to determine the apparent KD values.
The apparent dissociation constants were 37.9 plus or minus 1.0 micromolar for binding to the gas motif, and 23.3 plus or minus 0.6 micromolar for binding to the at rich oligonucleotide s plus 100. Surprisingly s plus 100 sequences displayed slightly tighter binding than gas. In three measurement repetitions, substitution of A to G resulted in a dramatic decrease in affinity of the s plus 100 mutant one.
While the s plus 100 mutant two showed no detectable binding, thus confirming sequence selective binding of STAT three to s plus 100 Once mastered, this technique can be done in less than two hours if it is performed properly. While attempting this procedure, it is important to remember that optimal conditions for extraction of membrane proteins will differ for different proteins and usually require optimization. After watching this video, you should have a good understanding of how to determine a binding affinity of a protein to a elegant without purifying the protein from the cell.Lysate.
