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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+polarization+assays+in+small+molecule+screening.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+biomolecules+using+surface+plasmons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+binding+assays+at+equilibrium:+validation+and+interpretation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+isothermal+titration+calorimetry+in+pure+and+applied+research--survey+of+the+literature+from+2010.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-protein+binding:+a+critical+review+of+analytical+tools.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+analytical+ultracentrifugation+in+protein+science:+a+tutorial+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+molecules+move+along+a+temperature+gradient.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+as+a+sensitive+method+to+quantify+protein:+nucleic+acid+interactions+in+solution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK-STAT+pathway+at+twenty.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+binding+specificity+of+different+STAT+proteins.+Comparison+of+in+vitro+specificity+with+natural+target+sites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+unphosphorylated+STATs+in+signaling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+unphosphorylated+STAT3+transcription+factor+binding+to+DNA.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+suppresses+transcription+of+proapoptotic+genes+in+cancer+cells+with+the+involvement+of+its+N-terminal+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+spectra.">Fluorescent protein spectra.</a> J. Cell Sci. 114, 837-838 (2001).</li></ol>

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 border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent/material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>RIPA buffer</td>
 <td>Millipore</td>
 <td>20-188</td>
 <td>Other manufacturer'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</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

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...

Watch this Scientific Journal Video about Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis at JoVE.com

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

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 ...

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30.3K Views.  National Cancer Institute. 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.

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

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...