Name: Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis (Video) | JoVE Journal
Uploaded: 2013-08-15T13:00:00.000+00:00
Duration: 10 min 22 s
Description: Learn microscale thermophoresis for binding affinity determination using GFP-fused proteins in cell lysates. This purification-free method streamlines quantitative characterization of protein-ligand interactions.

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This work was partially supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research; Collaborative Research Agreement between NCI and Calidris Therapeutics; American Cancer Society grant IRG 97-152-17 to O.T; and federal funds from the National Cancer Institute, NIH, under contract HHSN26120080001E.

1. Preparation of Cell Lysate
This protocol is intended for adherent cells expressing any GFP-fused protein. Needed cell number can vary from as low as 106 to as many as 20 x 106 cells, depending on the level of protein expression. For example, lysate of HEK cells overexpressing GFP-STAT3 was prepared by treating cells grown in 10 T75 flasks to near 70% confluency with 1 ml lysis buffer. However, this lysate had to be diluted 150-fold to provide optimal level of fluorescence for MST experiment. Cell lysis protocol depends strongly on the properties and intracellular localization of the protein under investigation. If using detergents is undesirable because of protein instability, sonication described below, could be the best choice. Different additives can be added to the lysis buffer to prevent protein modification reactions: EDTA prevents phosphorylation, sodium vanadate inhibits tyrosine protein phosphatases, sodium fluoride is an inhibitor of Ser/Thr phosphatases.
<ol>
	<li>Prepare lysis buffer. For easy-to-extract cytoplasmic proteins the following composition works well: 25 mM Tris HCl, pH 8.0, and protease inhibitor cocktail diluted 100-fold (Sigma-Aldrich P2714, consisting of 2 mM AEBSF, 0.3 &mu;M aprotinin, 130 &mu;M bestatin, 1 mM EDTA, 14 &mu;M E-64, 1 &mu;M leupeptin). Alternatively, for less soluble proteins, one can use commercial RIPA buffer with protease inhibitors cocktail and non-denaturing detergents. Keep the buffer on ice.</li>
	<li>Wash cells briefly with ice-cold PBS using 10 ml of buffer per T75 flask. Keep cells on ice for 5 min, or until cells start detaching from the flask. Scrape cells with cell scraper to detach if necessary.</li>
	<li>Resuspend cells in 10 ml ice cold PBS and transfer to a prechilled 14 ml round bottom centrifuge tube.</li>
	<li>Pellet cells by centrifugation at 400-600 x g at 4 &deg;C for 5 min. Combine cells from several flasks at this point if needed.</li>
	<li>Remove PBS supernatant and resuspend pellet in 200 &mu;l of ice-cold lysis buffer, transferring suspension to a pre chilled 1.5 ml Eppendorf tube.</li>
	<li>Keep cells on ice to minimize local overheating. Lyse cells with 3x 10 sec pulses of sonication at 30% amplitude, using a 2-3 mm pre-chilled tip. Keep the tip below the surface to minimize frothing. Omit this step when using detergent-containing buffer and incubate on ice for 30 min instead.</li>
	<li>Correct the lysate solution to contain physiological salt concentration (100 mM NaCl) if necessary using 5 M NaCl.</li>
	<li>Collect lysates by centrifugation at about 26,000 x g at 4 &deg;C for 10 min.</li>
	<li>Determine the optimal lysate dilution (as described in section 3 below) and amount of lysate needed for one titration (typically around 300 &mu;l of pre-diluted lysate).</li>
	<li>Aliquot the lysate and store at temperature appropriate for the protein under investigation (-80 &deg;C, in most cases).</li>
</ol>
2. MST Buffer Selection and Preparation
<ol>
	<li>Since the protein-ligand interactions are dependent on buffer conditions, MST buffer composition is chosen based on the properties of a particular system. It is generally advantageous to test at least two different buffers.</li>
	<li>Prepare 2 5x MST buffers. We had good experiences with these two compositions: HEPES (250 mM HEPES, pH 7.4; 25 mM MgCl2; 500 mM NaCl; 0.25% NP-40) and Tris HCl (250 mM Tris HCl, pH 7.4; 750 mM NaCl; 50 mM MgCl2; 0.05% Tween-20). Addition of BSA (5% in the final binding mixture) can help prevent adhesion of proteins to plastic tubes and glass capillaries. NaN3 (0.5 mM) can also be included to prevent growth of microorganisms.</li>
</ol>
3. Determination of Optimal Lysate Dilution
<ol>
	<li>Select the LED excitation source with the wavelength &lambda; = 470 nm on the MST instrument.</li>
	<li>Load capillaries with the cell extract diluted 2 and 10x with MST buffer.</li>
	<li>Perform "Find Capillaries" operation on Control Software of MST instrument. The optimal fluorescence range in diluted lysate is from 400 to 1,500 fluorescence units.</li>
</ol>
4. Determination of the Optimal Ligand Concentration Range
<ol>
	<li>The highest concentration of the ligand should be at least 20x higher than the expected dissociation constant.</li>
	<li>The lowest ligand concentration should be lower than molar concentration of the fluorescent protein.</li>
</ol>
Refer to the NanoTemper Technologies Concentration Finder tool for the ligand concentration range estimation.
5. Preparation of Cell Lysate and Ligand Dilutions
<ol>
	<li>Place a tube rack with a needed number (usually 10-16) of 0.5 ml LoBind centrifuge tubes on ice. Pipette 25 &mu;l of MST buffer to the bottom of each tube. Add 25 &mu;l of the ligand stock solution to the first tube (#1, ligand highest concentration) and perform serial two-fold dilution of the ligand using the rest of the tubes. Keep the rack with ligand samples on ice.</li>
	<li>Thaw cell lysate slowly on ice.</li>
	<li>Dilute cell lysate with MST buffer to provide the optimal level of the fluorescent target protein in the binding reactions. Final protein concentration should be close to expected KD or lower. It should be adjusted to obtain necessary number of fluorescence counts in the final solution. For determination of GFP-STAT3 concentration in the lysate, the instrument was calibrated with a help of fluorescein. The molar concentration of GFP in the lysate was determined using the ratio of fluorescein and EGFP quantum yields, 0.85 and 0.61 respectively.15</li>
</ol>
6. Microscale Thermophoresis Binding Studies
<ol>
	<li>Select the LED excitation source with &lambda; = 470 nm on the MST instrument.</li>
	<li>Place 0.5 ml LoBind tubes in the tube rack across from tubes with ligand serial dilution samples. Carefully add 15 &mu;l of the cell lysate to the bottom of each tube. Try not to touch tube walls to avoid sample loss.</li>
	<li>Add 15 &mu;l of the ligand sample with the highest concentration (#1) to the corresponding tube #1 with the cell lysate. Mix well and change a pipette tip. Repeat this step with the rest of the tubes except for the last one, which should contain no ligand.</li>
	<li>Add 15 &mu;l of MST buffer to the last tube and mix well.</li>
	<li>Fill approximately 2/3 of the first capillary with the binding mixture from tube #1, tilt it to move the solution towards the center, and place the capillary on the capillary tray to the position #1 (the closest position to the tray opening). Repeat this step with the rest capillaries. Capillary ends can be plugged with wax for longer experiments.</li>
	<li>Place the tray inside the MST instrument and close the instrument's door.</li>
	<li>Perform "Find Capillaries" command to let the instrument find exact positions of the capillaries and measure fluorescence of the samples.</li>
	<li>Based on the fluorescence signal intensity, adjust the LED power (from 10-100%) to bring it into 400-1,500 units interval.</li>
	<li>Click the "Start" button to perform the thermophoresis experiment. More than one IR-laser power can be chosen for the experiment in order to find the optimal temperature gradient for the particular system. Collect data from 2-3 runs for the same set of capillaries to ensure the reproducibility of measurements. It takes 10-12 min to run one set of 16 capillaries.</li>
</ol>
7. Microscale Thermophoresis Data Analysis
<ol>
	<li>Open the Analysis software.</li>
	<li>Load the project folder. In the appeared Information Run Viewer select collected at a specific IR laser power thermophoretic curves. There is an option of opening all thermophoretic traces collected under various conditions such as IR laser power, LED power, temperature, concentration, etc. at once and then choosing any curves for analysis by switching them on and off (click on the experiment's name).</li>
	<li>In the Evaluation Points graph window, select the thermophoresis or thermophoresis with T-jump. Ensure that blue and red lines are positioned correctly. To obtain the averaged points with standard deviations, select "Use Average" or "Distinguish runs" for separate runs.</li>
	<li>To plot a dissociation constant fit, select "Use Average", enter and fix the labeled molecule concentration value (check the "concentration" square in a fit window menu), and fit the curve. The KD value with its standard deviation appears in a separate information pop-up window. To plot a fit using Hill method, select "Average", Hill method, and then fit the curve. The EC50 affinity value with its standard deviation is shown in this case. The feature of KD or Hill "border" can also be utilized when saturation in a bound state is not reached.</li>
	<li>Save the average fit data in a text file and transfer to Excel.</li>
	<li>Plot Fnorm (normalized fluorescence), &Delta;Fnorm (difference in normalized fluorescence if different experiments are compared), or fraction bound (see the formula below) as a function of the unlabeled (titrated) molecule concentration.</li>
</ol>
Fraction Bound (fraction of molecules in a complex) = (Therm(C)-unbound)/(bound-unbound), where Therm(C) is thermophoresis measured for the concentration C, unbound is thermophoresis for the unbound state (when molecules are not in a complex), and bound is thermophoresis for the completely bound state.

<ol><li>Lea, W. A., Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+polarization+assays+in+small+molecule+screening%5BTitle%5D)+AND+%22Expert.+Opin.+Drug+Discov%22%5BJournal%5D)">Fluorescence polarization assays in small molecule screening.</a> Expert. Opin. Drug Discov. 6, 17-32 (2011).</li>
<li>Willander, M., Al-Hilli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+biomolecules+using+surface+plasmons%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Analysis of biomolecules using surface plasmons.</a> Methods Mol. Biol. 544, 201-229 (2009).</li>
<li>Hulme, E. C., Trevethick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ligand+binding+assays+at+equilibrium%3A+validation+and+interpretation%5BTitle%5D)+AND+%22Br.+J.+Pharmacol%22%5BJournal%5D)">Ligand binding assays at equilibrium: validation and interpretation.</a> Br. J. Pharmacol. 161, 1219-1237 (2010).</li>
<li>Ghai, R., Falconer, R. J., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+of+isothermal+titration+calorimetry+in+pure+and+applied+research--survey+of+the+literature+from+2010%5BTitle%5D)+AND+%22J.+Mol.+Recognit%22%5BJournal%5D)">Applications of isothermal titration calorimetry in pure and applied research--survey of the literature from 2010.</a> J. Mol. Recognit. 25, 32-52 (2012).</li>
<li>Vuignier, K., Schappler, J., Veuthey, J. L., Carrupt, P. A., Martel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug-protein+binding%3A+a+critical+review+of+analytical+tools%5BTitle%5D)+AND+%22Analytical+and+Bioanalytical+Chemistry%22%5BJournal%5D)">Drug-protein binding: a critical review of analytical tools.</a> Analytical and Bioanalytical Chemistry. , 398-3953 (2010).</li>
<li>Lebowitz, J., Lewis, M. S., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modern+analytical+ultracentrifugation+in+protein+science%3A+a+tutorial+review%5BTitle%5D)+AND+%22Protein+Sci%22%5BJournal%5D)">Modern analytical ultracentrifugation in protein science: a tutorial review.</a> Protein Sci. 11, 2067-2079 (2002).</li>
<li>Duhr, S., Braun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+molecules+move+along+a+temperature+gradient%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Why molecules move along a temperature gradient.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 19678-19682 (2006).</li>
<li>Zillner, K., Jerabek-Willemsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscale+thermophoresis+as+a+sensitive+method+to+quantify+protein%3A+nucleic+acid+interactions+in+solution%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution.</a> Methods Mol. Biol. 815, 241-252 (2012).</li>
<li>Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">Protein-binding assays in biological liquids using microscale thermophoresis.</a> Nat. Commun. 1, 100(2010).</li>
<li>Stark, G. R., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+JAK-STAT+pathway+at+twenty%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The JAK-STAT pathway at twenty.</a> Immunity. 36, 503-514 (2012).</li>
<li>Ehret, G. B., Reichenbach, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+binding+specificity+of+different+STAT+proteins.+Comparison+of+in+vitro+specificity+with+natural+target+sites%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.</a> J. Biol. Chem. 276, 6675-6688 (2001).</li>
<li>Yang, J., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+unphosphorylated+STATs+in+signaling%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">Roles of unphosphorylated STATs in signaling.</a> Cell Res. 18, 443-451 (2008).</li>
<li>Timofeeva, O. A., Chasovskikh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+unphosphorylated+STAT3+transcription+factor+binding+to+DNA%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Mechanisms of unphosphorylated STAT3 transcription factor binding to DNA.</a> J. Biol. Chem. 287, 14192-14200 (2012).</li>
<li>Timofeeva, O. A., Tarasova, N. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAT3+suppresses+transcription+of+proapoptotic+genes+in+cancer+cells+with+the+involvement+of+its+N-terminal+domain%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain.</a> Proc. Natl. Acad. Sci. U.S.A. , (2013).</li>
<li>Patterson, G., Day, R. N., Piston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+protein+spectra%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Fluorescent protein spectra.</a> J. Cell Sci. 114, 837-838 (2001).</li>
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The authors declare that they have no competing financial interests. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Protein expression and purification is a laborious and expensive step, which is, however, necessary for determination of interactions' KD by most currently used method. Application of MST allows avoiding protein purification thus significantly simplifying and accelerating quantitative characterization of interactions. It presents particularly significant advantages in the case of difficult-to-express and purify proteins, such as membrane proteins and transcription factors.
The major...

Quantitative characterization of affinity of intermolecular interactions is important in many areas of biomedical research. Binding dissociation constant (KD) is essential not only in drug discovery but is also an important parameter in characterization of any binary interaction in any biological system. Biochemical methods used for detection of protein-protein interactions, such as immunoprecipitation and yeast two-hybrid screens, do not inform us on how tight are those interactions, while affinity defines whether this particular complex exists under given conditions in vivo. In drug discovery process, binding assay development is one of the necessary and frequently the most time-consuming steps. Most commonly used methods of KD determination include fluorescence polarization,1 surface plasmon resonance (SPR) technology,2 radioligand binding,3 isothermal titration calorimetry,4 equilibrium dialysis (ED),5 ultrafiltration (UF),5 and ultracentrifugation (UC).6 All of them require significant quantities of purified target protein. Microscale thermophoresis (MST) is a rapidly developing method that detects directed movement of molecules in a microscopic temperature gradient. Any changes of the hydration shell of biomolecules result in a relative change of movement along the temperature gradient.7 MST is used to determine binding affinities and has been applied for studying ligand binding to fluorescently labeled proteins or fluorescent ligands to a target protein.8, 9 MST allows measurement of interactions directly in solution without the need of immobilization to a surface (immobilization-free technology). Practically, any binding is accompanied by a change in MST signal, although the size of the change differs from system to system significantly. For the detection of molecule motion by MST, they have to be fluorescent. This major limitation of the method can be turned into an advantage. If a protein is expressed as a GFP fusion in any system, it will be the only fluorescent molecule and thus can be studied without isolation from the cell lysate or cell-free expression system. Generation of cell lysates that allow for binding conditions with minimal artifacts is the major challenge. Here we describe a protocol of cell lysate preparation and MST experiment that can be used for many soluble and membrane proteins.
STAT proteins are latent cytoplasmic transcription factors activated by tyrosine phosphorylation in response to extracellular signals and are involved in many biological processes including immunity, hematopoiesis, inflammation, and development.10 In mammals, the STAT family consists of STAT 1, 2, 3, 4, 5A, 5B, and 6. All activated STATs are known to bind to the same DNA sequence, so called GAS motif, IFN-gamma activated sequence. However, the transcriptional effects of different STATs are very different.11 In spite of involvement in many pathological processes and extensive studies yielding over 17,000 publications, the KD of STAT interactions with different DNA sequences have not been determined. Only relative affinity of different STATs to variants of GAS motif has been characterized.11 Difficulties in protein expression and purification are the major impediments in characterization of STATs' DNA binding selectivity. Although the majority of the studies have focused on the role of "activated" STATs, which became synonymous to Tyr-phosphorylated transcription factor, the role of non-phosphorylated STATs (U-STATs) in regulation of transcription is emerging rapidly.12 However, these mechanisms are poorly understood, and it was unclear whether U-STATs actually bind to DNA or act through interactions with other transcription factors. We have recently shown that U-STAT3 can bind to DNA sequences different from GAS motifs with even higher affinity.13 The finding has significant implications for our understanding of the biological functions of this important protein. We have applied microscale thermophoresis to determine relative affinities of STAT3 to GAS and AT-rich oligonucleotide S+100 (Figure 4). Almost identical protocol has been used for KD determination for binding of a different STAT3 ligand, a lipopeptide inhibitor.14 No binding to a related transcription factor, GFP-STAT1 that was used as a negative control could be detected thus confirming selectivity of interaction.14

<table><tbody><tr><td>RIPA buffer</td><td>Millipore</td><td>20-188</td><td>Other manufacturer&#039;s buffers work as well</td></tr><tr><td>Protease inhibitors cocktail</td><td>Sigma-Aldrich</td><td>P2714</td><td></td></tr><tr><td>Monolith NT.115</td><td>NanoTemper Technologies GmbH</td><td>G008</td><td></td></tr><tr><td>Monolith NT.115 Capillary Tray</td><td>NanoTemper Technologies GmbH</td><td>T001</td><td></td></tr><tr><td>Monolith NT.115 Standard Treated Capillaries</td><td>NanoTemper Technologies GmbH</td><td>K002</td><td></td></tr><tr><td>NT Control software</td><td>NanoTemper Technologies GmbH</td><td>2.0.2.29</td><td></td></tr><tr><td>NT Analysis software</td><td>NanoTemper Technologies GmbH</td><td>1.4.27</td><td></td></tr><tr><td>Table-top refrigerated centrifuge</td><td>Eppendorf</td><td>5417R</td><td>Other microtube refrigerated centrifuges providing </td></tr><tr><td>Protein LoBind Tube 0.5 ml</td><td>Eppendorf</td><td>22431064</td><td></td></tr></tbody></table>

protein purification-free method of binding affinity determination by microscale thermophoresis

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Measuring the affinity of non-phosphorylated STAT3 protein binding to oligonucleotides.
HEK293 cells expressing STAT3-GFP were used as a source of fluorescently labeled STAT3 for DNA binding assay. Cell lysates were prepared using RIPA buffer (20x106cells/ml). For binding studies, the lysates were diluted 150x with MST DNA-binding buffer to provide the optimal level of the fluorescent protein in the binding reaction (about 20 nM). Non-transfected HEK293 cells have been...

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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.

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ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

This video demonstrates microscale thermophoresis for studying the protein-lipid interaction. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent-labeled proteins in response to a temperature gradient. The fluorescent molecule is mixed with different concentrations of the non-fluorescent lipid molecules, and the mixture of molecules in the solution is loaded into capillaries. A temperature gradient is applied to the samples in the...

Microscale Thermophoresis to Study Protein-Lipid Interactions in Solution

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Learn microscale thermophoresis for binding affinity determination using GFP-fused proteins in cell lysates. This purification-free method streamlines quantitative characterization of protein-ligand interactions.

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

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