This work was supported by the Canadian Institutes of Health Research (CIHR), National Science and Engineering Research Council (NSERC), and a CIHR training grant scholarship (to L.F.). We thank Prof. Gerard D. Wright (McMaster University, Canada) for the AAC(6)-Ii expression plasmid.

1. Preparing stock solutions
<ol>
	<li>Purify the macromolecule of interest. (In this case, aminoglycoside N-6'-acetyltransferase-Ii (AAC6'-Ii), is isolated as reported elsewhere.13)</li>
	<li>Prepare 4 litres of dialysis buffer. (In this case we used 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, MW 238.3 g/mol), containing 2 mM ethylenediaminetetraacetic acid (EDTA, MW 292.2), at pH 7.5.)</li>
	<li>Dialyze the protein The AAC(6')-Ii sample (5 mL at 400 &mu;M) used must be dialyzed in dialysis buffer (3 x 1.3 L). The final dialysis solution is kept to rinse the machine and to dilute samples.</li>
	<li>Filter the final dialysis solution through a 0.45 &mu;m cellulose filter, and stored at 4&deg;C. Will be referred to as 'running buffer'</li>
	<li>Filter protein solution through a 0.2 &mu;m syringe filter which has been rinsed thoroughly with running buffer. Measure the final concentration of the protein using a standard assay (Bradford, Lowry, etc.) or UV absorbance. Store the protein to maximize long-term stability. (In the case of AAC(6')-Ii, this means storage at 4&deg;C. AAC(6')-Ii does not retain activity after freezing and thawing).</li>
	<li>Prepare 200&mu;L of 25 mM stock solution of acetyl coenzyme A (AcCoA, the ligand, MW 809.57) by dissolving 4.0 mg in running buffer. Freeze at -78&deg;C until ready to use.</li>
	<li>All protein and ligand samples must originate from the same stock solutions, to minimize random sample-to-sample fluctuations in concentration. A single correction factor for the protein concentration across the entire variable-c dataset is adjusted in the analysis.10</li>
</ol>
2. Preparing ITC samples
<ol>
	<li>Dilute the enzyme solution to a c-value of 64 with a final volume of 2 mL. (In the case of AAC(6')-Ii, this corresponds to 192 &mu;M.)</li>
	<li>Quickly thaw the ligand (AcCoA) stock solution in ice water.</li>
	<li>Prepare 0.5 mL of a AcCoA solution 10 times more concentrated then the number of binding sites on the protein, in this case 4 mM, by diluting 80 &mu;L of the AcCoA stock solution in 420 &mu;L of running buffer. Transfer into a pipette filling tube. Quickly return the AcCoA stock solution to -78&deg;C.</li>
	<li>Degas the protein and solutions under vacuum for 5 min at a temperature 1&deg;C below the desired running temperature (19 &deg;C).</li>
</ol>
3. Setting up the syringe14
<ol>
	<li>Insert the injection syringe through syringe holder until the pre-mounted syringe clamp is at the same height as the holder.</li>
	<li>Feed the second syringe clamp over the injection syringe until it is firmly pressed against the bottom of the syringe holder. Gently tighten the clamp with the provided 0.050" Ball Point Hex Drive.</li>
	<li>Place syringe holder into the pipette holder.</li>
	<li>Gently slide the pipette injector into the injection syringe. Make sure the plunger tip is fed directly into the hole of the syringe. Once fully inserted, screw the locking collar of the syringe holder into the pipette injector.</li>
</ol>
4. Loading the sample cell14
<ol>
	<li>Wash sample cell with a minimum of 50 mL of running buffer, and remove any remaining liquid by using a long-needled 2.5 ml glass syringe.</li>
	<li>Slowly draw a minimum of 1.8 ml of protein sample solution into the clean and dry long-needled 2.5 ml syringe. Be careful not to introduce any bubbles.</li>
	<li>Carefully insert needle in sample cell and gently touch the bottom of the cell. Raise tip slightly (~1 mm) and gently inject AAC(6')-Ii solution into the cell until excess liquid is visible above the top of the sample cell. </li>
	<li>Slowly raise the needle about 1 cm while ensuring liquid remains in the overflow. Quickly withdraw and inject a small amount of solution (~0.25 ml) to remove any trapped bubbles in the sample cell.</li>
	<li>Remove all solution overflow. This is achieved by gently sliding the needle along side of overflow into the sample cell. The syringe tip will hit a ledge, this is the desire height for the running solution. Remove all liquid which sits above this line.</li>
</ol>
5. Loading injection syringe and initiating run14
<ol>
	<li>Attach the plastic tube loading syringe into fill port. </li>
	<li>Lower plunger tip to the top of the fill port.</li>
	<li>Place the AcCoA solution in the pipette filling tube at the bottom of the pipette holder. The syringe tip should not touch the bottom of the filling tube.</li>
	<li>Slowly draw the solution into the syringe until a small amount enters the tube of the loading syringe.</li>
	<li>Close the fill port by lowering the plunger tip and click on the Close Fill Port button. Purge and refill, by clicking on the Purge->Refill button, 3 times to remove any air bubbles that may have become trapped during loading.</li>
	<li>Remove the filling tube from the pipette holder and gently wipe the syringe tip.</li>
	<li>Take pipette assembly and gently lower syringe into the sample cell. Proceed slowly as the syringe can easily bend, so great care is needed. Ensure the syringe is completely inserted by pressing down on base of locking collar.</li>
	<li>Set the desired running temperature (in this case 20&deg;C), and select a reference power that is slightly greater than the maximum injection heat flow expected (in this case 20&mu;cal/sec).</li>
	<li>Program the desired injection volumes and delays. (In this case, 28 injections were employed. The first injection had a volume of 2 &mu;L with a 60 s delay. All subsequent injections had volumes of 10 &mu;L with 330 s delays.)</li>
</ol>
6. Subsequent Runs
<ol>
	<li>In all subsequent runs repeat steps 2-5, while decreasing AAC(6')-Ii and AcCoA concentrations by a factor of 2,4,8,16, and 32.</li>
</ol>
7. Data Analysis
<ol>
	<li>Use a fitting procedure which globally fits all of the isotherms to a single set of binding parameters, as described previously10.</li>
</ol>
8. Representative Results
Representative data are shown in Figure 1. The shapes of the isotherms should vary with concentration. Sharper transitions are expected for higher c-values (i.e. higher protein and ligand concentrations) (Figure 2). 
In the case of AAC(6')-Ii, the two-site sequential model gives a better fit than one describing two sets of identical, independent sites with adjustable stoichiometries. 
<img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Figure 1" /> Figure 1. Isotherms produced by titration of AcCoA (3.86 mM) into AAC(6')-Ii (192 &mu;M). A) Raw ITC trace. B) Integrated values used for determining binding parameters (squares) with a 2-site sequential fit (-). 
<img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Figure 2" /> Figure 2. ITC isotherms for AcCoA titrated into AAC(6')-Ii at varying concentrations. The experimental data (open circles) were fit to a 2-sets-of-sites independent model (dashed magenta) and a 2-site sequential model (solid blue). The 2-site sequentional model clearly gives better overall agreement. The concentrations employed were A) 6 &mu;M, 0.25 mM, B) 12 &mu;M, 0.25 mM, C) 24 &mu;M, 0.5 mM, D) 48 &mu;M, 1.0 mM, E) 96 &mu;M, 1.9 mM, and F) 196 &mu;M, 3.86 mM, for AAC(6')-Ii and AcCoA respectively.

<ol>
	<li>Cliff, M. J., Ladbury, 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=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, 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=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, 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=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, 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=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., 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=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

No conflicts of interest declared.

This analytical portion of variable-c fitting has been previously described in detail10. Here we report practical aspects of collecting variable-c datasets suitable for this approach. It is essential that all protein and ligand samples are drawn from the same stock solutions. Therefore it is important that sufficient stock solution is prepared initially to complete the entire series of experiments. This ensures the ratio of AAC(6')-Ii and AcCoA is constant among all experiments, and reduces random fluctuations in concentration on a sample-to-sample basis.
In this case, much care must be taken in the handling of the ligand. AcCoA is chemically unstable in solution so it is essential that the stock solution remains liquid for very short periods of time. Once the appropriate amount is aliquoted from the stock solution, the stock must immediately be refrozen. If this is not done, then the AcCoA may degrade over time and the concentrations will not be correct in later runs. Once aliquoted, AcCoA samples must be used immediately. In the case of proteins that are unstable, similar precautions will be necessary.
This procedure can be easily modified to study other complex systems.10 The concentrations used must be adapted to the binding constants of the specific system. A wide range of c values between about 1 and 10008,10 is required across the dataset.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

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

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 ...

collecting-variable-concentration-isothermal-titration-calorimetry

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12.7K Views.  McGill University. 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.

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

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

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 ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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.

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 ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

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 ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...