This work was supported by NSF grant MCB-0817827.

Isothermal titration calorimetry (ITC) is a well established technique that can determine all the thermodynamic parameters (affinity, enthalpy and stoichiometry) of a binding interaction in one experiment.1 ITC works by titrating one reactant into a second reactant under isothermal conditions. The signal measured is the heat released or absorbed upon interaction (binding) of the two reactants. A series of injections are performed and the heat signal will approach zero as the limiting reactant becomes saturated. Fitting of the isotherm gives the thermodynamic parameters. Several reviews are available that describe the instrumentation as well as the math of data collection and analysis. 2,3 While other calorimeters are available (most notably the ITC200 with small volumes), here we describe a general protocol for the VP-ITC manufactured by MicroCal (now part of GE Healthcare).
1. Preparing Samples
<ol>
	<li>In order to prepare the macromolecule and ligand in buffer, some potential issues need to be addressed. Accurate fits require proper concentrations of the macromolecule, the species that typically goes in the sample cell of the ITC, and ligand, the species in the injection syringe. Because some proteins aggregate at the high concentrations needed for the species in the injection syringe, most often the protein is loaded into the sample cell. The optimal macromolecule concentration is determined from "c", the product of the predicted affinity of the system, which can be estimated using orthogonal methods prior to using ITC, and the total macromolecule concentration, where c = Ka*[M]. Optimal values of c range from 10-1000, 1 though it is possible to get accurate data for weak-binding systems under specific experimental conditions with c-values below the lower limit.4 Thus, the macromolecule concentration must be determined with this range of c values in mind (i.e., for a Ka of 106 M-1, macromolecule concentrations of 10 to 1000 &mu;M should be used). Prior knowledge of the binding affinity of the system can help minimize the protein used for ITC through better design of the ITC experiment. The concentration of the ligand should be large enough (7-25 fold more concentrated than the Kd for the weakest ligand binding site) so that saturation occurs within the first third to half of the titration. Accurate fitting of the data also requires saturation of the signal. For systems with higher binding affinity, a lower ligand concentration should be used to avoid saturation too early in the titration, which will give inaccurate fits. Once the heat of dilution control (i.e. titration of ligand into buffer) has been subtracted from the titration, the enthalpy at saturation should approach zero.</li>
	<li>Because small molecule impurities can give rise to artifactual signals in the ITC measurements, it is best, if possible, that the macromolecule and ligand be exhaustively dialyzed against buffer. Alternatively, column chromatography, desalting spin columns, or buffer exchange centrifugal filters (for example, Centricons) can be used to change the buffer of the macromolecule. If the ligand is a small molecule, it can be prepared using the dialysis buffer after the macromolecule has been dialyzed or by dialyzing against dialysis membranes with cutoffs suitable for small molecules (i.e. 100-500 Da for a Spectra/Por Float-A-Lyzer). Differences in the buffer composition between the ligand and macromolecule solutions can lead to signal artifacts from the heat of the dilution of impurities in the samples. After preparation, check to make sure the pH of the buffer, macromolecule and ligand match (&plusmn; 0.05 pH units) as artifacts in the enthalpy can arise due to buffer protonation effects.5 Make sure to prepare enough of each species. For triplicate experiments and a dilution control, the amount of material needed will depend upon the ITC used. But, for most ITC instruments that have approximate 2 ml volume sample cells, at least 6-7 ml of macromolecule solution will be needed, and can be conveniently prepared in 15 ml falcon tubes. For 300 &mu;L injection syringes, 1-2 ml of solution should be adequate, and can be prepared in either falcon tubes or microcentrifuge tubes.</li>
	<li>Dust, and other particulates, can cause artifacts in the baseline of the ITC thermogram. It is imperative that they be removed prior to running the experiment. After preparation of sample stock solutions, the samples should be centrifuged in microcentrifuge tubes for five minutes at 8000 to 14,000 RPM to pellet particles in the solution. Remove the supernatant, being careful not to disturb the pellet, and place it in a new falcon/microcentrifuge tube. 
	If reductants are needed to maintain reduced cysteines, use low concentrations and fresh stocks of &beta;-mercaptoethanol,TCEP (tris(2-carboxyl)phosphine) or dithiothreitol to minimize any artifacts due to reductant oxidation. Also, the presence of organic solvents in the buffer (common for some small molecule ligands that may need methanol or DMSO in order to be soluble) can cause signal artifacts. If organic solvents are needed for the ligand, then the macromolecule solution must also contain the same concentration to avoid any signal arising from the heat of dilution of the organic solvent. Slight differences in buffer composition between the ligand and macromolecule due to cosolvents, salts or pH are possible during preparation of the samples. It is best to check the dilution of the ligand into the sample buffer or the sample buffer into the macromolecule to ensure that heat signals arising from differences in the buffer content don't cause data arising solely from artifacts.</li>
	<li>Check the concentrations of the macromolecule and ligand carefully using techniques suitable to your system (such as absorbance measurements, HPLC ,colorimetric assays, BCA assays for proteins, etc) to record their exact concentrations. Differences in the actual concentration and the concentration used to fit the isotherm will cause errors in the stoichiometry, enthalpy and binding affinity determined from the experiment. It is common to degas the samples to avoid signal artifacts due to air bubbles or release of dissolved gases during the titration, particularly at higher temperatures.</li>
</ol>
2. Setting up the Experiment
<ol>
	<li>Make sure the sample cell and injection syringe are cleaned according to the manufacturer's protocol prior to loading the macromolecule and ligand. Rinse the sample cell two or three times with 1.8 ml of distilled water using the Hamilton syringe (use care with the Hamilton syringe as the barrel is easily broken or the needle bent). Next rinse the sample cell several times with 1.8 ml of buffer. Load the sample cell with 1.8 ml of macromolecule solution, being careful to avoid bubble formation.</li>
	<li>Fill the reference cell with distilled water. For most buffers, distilled water is fine to use as the reference solution. However, for buffers with particularly high ionic strengths or osmolality, it is better to use the buffer as a reference.</li>
	<li>Remove air bubbles from the reference and sample cells using the Hamilton syringe. Gently move the needle of the syringe up-and-down the sides of the cell knocking any bubbles that may be at the bottom of the cell and attached to the well to the top of the cell. Remove any excess volume from the sample and reference cells.</li>
	<li>Attach a plastic syringe to the fill port of the injection syringe using tubing. Rinse the injection syringe with distilled water. Follow by a buffer rinse. Make sure the injection syringe is completely evacuated by drawing air through the system. Place the needle of the injection syringe into the ligand solution and draw the ligand solution into the injection syringe until the entire syringe is full. Continue drawing a little excess volume (approximately 50 &mu;l or more) into the port and attached tubing. Immediately close the fill port of the syringe and detach the tubing and plastic syringe. Purge and refill the injection syringe two more times to remove any bubbles from the syringe. Remove the syringe from the ligand solution and wipe the side with a kimwipe to remove any drops, being careful not to touch the syringe tip to the kimwipe as this may remove volume from the syringe. Also, be careful not to knock or jar the syringe as this also can cause loss of volume from the syringe tip. Place the injection syringe into the sample cell.</li>
	<li>Set up the parameters for running the ITC. For binding systems with strong heat signals, a large number of low volume injections will give more data points for fitting (e.g. 75 injections of 3 &mu;l). For systems that have weak heat signals, a small number of large volume injections are preferable (e.g. 33 injections of 8 &mu;l). Most commonly, fewer injections with higher injection volumes are used. It may take several titrations to optimize the conditions that are best for your system. It is important to note that the binding enthalpy can either be exothermic or endothermic, depending upon the system being studied. Unfortunately, some systems have low heat signals, making the heat of reaction difficult to determine. Problems with such systems can potentially be overcome by increasing the concentration of the macromolecule, changing the temperature of the experiment (depending upon the heat capacity of the binding system), and/or altering the pH or ionic strength of the buffer.</li>
	<li>Also consider the time spacing between each injection. It is imperative that after each injection of ligand, the system is given time to equilibrate and the heat signal returns to baseline before the next injection occurs. For most systems, three to five minutes should be adequate. The time between injections should be increased for systems where the equilibration doesn't occur within five minutes.</li>
	<li>Because there will be some mixing between the macromolecule and ligand solutions in the injection syringe once it is inserted into the sample cell, the first injection will give spurious results. It is best to use small volumes (e.g. 2 &mu;l) for the first one or two injections (so they can later be discarded) and keep the subsequent injections at the desired values.</li>
	<li>Choose the temperature of the experiment (with 25 &deg;C being the most common, though temperatures between 2 and 80 &deg;C can be used). It is best to choose a temperature that matches other experiments (binding, kinetics, etc.) performed on the ligand-macromolecule system. The ITC can be set up to equilibrate at a temperature different from the temperature of the experiment. If the experiment is going to be performed at a temperature more than 10 &deg;C away from room temperature, then it is best to set the equilibration temperature for the instrument within 5 &deg;C of the experimental temperature. This will decrease the time the instrument will take to reach the temperature of the experiment. 
	The stirring speed of the syringe also needs to be considered. Stirring is necessary for adequate mixing of the ligand and macromolecule during the titration, but some proteins are destabilized by rapid stirring. For these cases, the stirring speed should be set at a relatively low rate. Once all the experimental parameters have been set up, then the experiment can be started.</li>
	<li>Once the experiment has finished, the ITC can be cleaned according to the manufacturer's protocol. The solution in the sample cell at the end of the experiment can be kept if additional experiments on the macromolecule-ligand complex mixture are desired.</li>
	<li>Repeat the titrations at least one or two more times to get reproducible data. Run a control where ligand is titrated into buffer in the sample cell to determine the heat of dilution for the ligand. For some systems where there is cooperative binding, additional information about the binding process can be gained by injecting the macromolecule into the ligand. The different injection orientations can give additional information, which may be helpful for global fitting.6</li>
</ol>
3. Analyzing the Data
<ol>
	<li>Fitting of the data can be easily performed using macros in any data fitting program (usually supplied by the manufacturer along with the instrument). Load the first data file. Check the raw thermogram for any signs of air bubbles or other artifacts in the signal. If there are any artifacts (such as spikes in the baseline or peaks when there was no injection at that time point), then note these data points as they should be removed. Next, load the ligand dilution control data, and subtract the ligand dilution data from the binding isotherm. At this time, remove all spurious data points, including the first one or two data points of the titration where dilution artifacts occur (refer to step 7).</li>
	<li>Select the data fitting model (one binding site, two/multiple binding sites, cooperative binding, etc) to be used to fit the data. The data can be fit with initial guesses of the fitting parameters, stoichiometry (n), enthalpy (&Delta;H) and binding affinity (Ka). If prior knowledge of the binding affinity, or other parameters, is known from orthogonal experiments, then these values can be entered. This helps avoid the fit becoming trapped in local minima during fitting. Once the data have been fit for the first titration, repeat steps 15 and 16 for the rest of the isotherms. Alternatively, the data can be fit globally using programs such as SEDPHAT.6 SEDPHAT may be particularly helpful in global fitting of binary complex datasets (i.e. molecules A+B) in combination with ternary complex datasets (molecules A+B+C). For example, in binding of molecule C to an A-B complex, it is not always clear whether there might be any signal due to binding of C to A or of B to A (if A is not fully saturated).</li>
	<li>To ensure that the ITC data are not artifactual, the binding affinity and stoichiometries obtained from ITC should be compared with an orthogonal method.7,8 Additionally, the ITC enthalpy value can be compared to the enthalpy from a van't Hoff plot.9 It can also prove helpful to use different concentrations of the ligand or macromolecule as the absolute value of the heat signal should increase with increasing macromolecule concentration. Artifacts are most likely to arise if the buffers in the cell and syringe are not matched. Another possibility for artifacts arises from impure samples.</li>
	<li>We recommend that the user start with simple binary complex titrations for which there is previous information available on Kd and stoichiometry. Then, if additional ligands bind, the user can try more complicated ternary complex titrations, varying the ligand concentrations, and perhaps switching the syringe and cell components. A comparison of enthalpies for both paths to ternary complex formation should be additive as enthalpy is a state function.10 Again, SEDPHAT6 is likely to be quite useful here.</li>
</ol>
4. Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/2796/2796fig1.jpg" /> 
Figure 1. A representative example of a well-behaved titration for the binding of the cofactor NADPH to E. coli chromosomal dihydrofolate reductase (ecDHFR). Panel (A) shows the raw thermogram; (B), the binding isotherm from the integrated thermogram fit using the one-site model in the Origin software; and (C) the fit of the isotherm using the one binding site model from SEDPHAT along with fit residuals. From the Origin software, using the one site binding model, n = 1.09 &plusmn; 0.02, Kd = 0.194 &plusmn; 0.001 &mu;M, &Delta;H = -22.7 &plusmn; 0.4 kcal/mol, T&Delta;S = -13.1 &plusmn; 0.4 kcal/mol, and &Delta;G = -9.16 &plusmn; 0.01 kcal/mol. Fits of the data using Sedphat afford an n of 0.94 &plusmn; 0.01, a Kd of 0.195 &plusmn; 0.013 &Delta;M, a &Delta;H of -22.5 &plusmn; 0.2 kcal/mol, T&Delta;S of -13.39 kcal / mol and &Delta;G of -9.15 kcal/mol.

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+binding+stoichiometries+and+cooperativity+are+observed+during+binary+and+ternary+complex+formation+in+the+single+active+pore+of+R67+dihydrofolate+reductase,+a+D2+symmetric+protein.">Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein.</a> Biochemistry. 35, 11414-11424 (1996).</li><li>Shenoy, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+and+multivalent+binding+between+cyanovirin-N+and+branched+oligomannosides:+calorimetric+and+NMR+characterization.">Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.</a> Chem. 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J., Haq, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+analysis+of+isothermal+titration+calorimetry+data+for+ligand-DNA+interactions.">Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions.</a> Methods. 42, 162-172 (2007).</li><li>Salim, N. N., Feig, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+of+RNA.">Isothermal titration calorimetry of RNA.</a> Methods. 47, 198-205 (2009).</li><li>De, M., You, C. C., Srivastava, S., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+interactions+of+proteins+with+functionalized+nanoparticles:+a+thermodynamic+study.">Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study.</a> J. Am. Chem. Soc. 129, 10747-10753 (2007).</li><li>Velazquez-Campoy, A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+to+determine+association+constants+for+high-affinity+ligands.">Isothermal titration calorimetry to determine association constants for high-affinity ligands.</a> Nat. Protoc. 1, 186-191 (2006).</li><li>Velazquez Campoy, A., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITC+in+the+post-genomic+era...?+Priceless.">ITC in the post-genomic era...? Priceless.</a> Biophys. Chem. 115, 115-124 (2005).</li><li>Harries, D., Rau, D. C., Parsegian, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solutes+probe+hydration+in+specific+association+of+cyclodextrin+and+adamantane.">Solutes probe hydration in specific association of cyclodextrin and adamantane.</a> J. Am. Chem. Soc. 127, 2184-2190 (2005).</li><li>Chervenak, M. C., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+measure+of+the+contribution+of+solvent+reorganization+to+the+enthalpy+of+binding.">A direct measure of the contribution of solvent reorganization to the enthalpy of binding.</a> J. Am. Chem. Soc. 116, 10533-10539 (1994).</li><li>Taneva, S. G., Moro, F., Velazquez-Campoy, A., Muga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+nucleotide-induced+DnaK+conformational+states.">Energetics of nucleotide-induced DnaK conformational states.</a> Biochemistry. 49, 1338-1345 (2010).</li><li>Jelesarov, I., Bosshard, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+ferredoxin+binding+to+ferredoxin:NADP++reductase+and+the+role+of+water+at+the+complex+interface.">Thermodynamics of ferredoxin binding to ferredoxin:NADP+ reductase and the role of water at the complex interface.</a> Biochemistry. 33, 13321-13328 (1994).</li><li>Burrows, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+monomer-dimer+equilibrium+of+interleukin-8+reveals+it+is+a+monomer+at+physiological+concentration.">Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentration.</a> Biochemistry. 33, 12741-12745 (1994).</li><li>Baker, B. M., Murphy, K. 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No conflicts of interest declared.

ITC has been used extensively in studying ligand macromolecule interactions,11 with studies looking at protein-ligand,12 DNA-ligand13 and RNA-macromolecule14 
studies. ITC can even be run with solid materials, such as nanoparticles, that form uniform suspensions.15 Further, ternary systems, where one ligand is already bound to the macromolecule and a second ligand is titrated, can be used to determine the thermodynamics of, for example, binding of substrate to an enzyme-cofactor system.7 Studies can also be performed for molecules with very high binding affinities that would normally exceed the detection limit of the ITC by performing competition binding assays with weaker binding ligands.16 Information on weakly binding ligands can also be obtained by competition assays.17 The role of water in binding can be explored by ITC,18 along with the dependence of the enthalpy on solvent reorganization.19 Recently, the thermodynamics of conformational change of DNAK variants were measured upon binding of ADP and ATP.20 Protein-protein association can be studied by ITC, yielding information on hetero complexes21 as well as on homo-association.22 Temperature effects on the binding enthalpy will give the heat capacity of the binding event.2 The number of protons absorbed or released upon binding can also be determined from experiments performed in buffers with different enthalpies of ionization.5 If additional ITC studies are done at different pH values, then the pKa of the group involved can potentially be determined. 23
To summarize, accurate measurements of the concentration of the macromolecule and ligand are imperative for a good ITC experiment. Sample concentrations also need to be within a proper range to get reliable data. Care should be taken with the buffer as small molecule impurities and pH mismatches will cause artifacts in the thermogram.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>1.7 mL microcentrifuge tubes</td>
 <td>Fisher Scientific</td>
 <td>02-681-282</td>
 </tr>
 <tr>
 	<td>15 mL falcon tubes</td>
 <td>Fisher Scientific</td>
 <td>14-959-49B</td>
 </tr>
 <tr>
 	<td>2.5 mL Hamilton syringe</td>
 <td>MicroCal</td>
 <td>SYN161714</td>
 </tr>
 </tbody>
</table>

isothermal titration calorimetry for measuring macromolecule-ligand affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

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Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

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 (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

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52.7K Views.  University of Tennessee. Un protocollo generale per l'uso di calorimetria isotermica di titolazione per monitorare la termodinamica vincolante per i sistemi biologici con moderata affinità di legame è presentato.

Video: Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

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

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

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.

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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 ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...