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 ...

<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>

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 fluctuation...

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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 ビュー.  McGill University. ITCは、そのホストへのリガンドの結合を研究するための強力なツールです。複雑なシステムではしかし、いくつかのモデルも同様にデータをフィットすることがあります。ここで説明する方法は、複雑なシステムのための適切な結合モデルを解明し、対応する熱力学的パラメータを抽出する手段を提供します。

ビデオ: 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...

isothermal titration calorimetry for measuring macromolecule-ligand affinity

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.

hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean...

measuring enzymatic stability by isothermal titration calorimetry

Measuring Enzymatic Stability by Isothermal Titration Calorimetry

The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme...

isothermal titration calorimetry for evaluating protacs-induced ternary complex formation

Isothermal Titration Calorimetry for Evaluating PROTACS-Induced Ternary Complex Formation  

determining the thermodynamic and kinetic association of a dna aptamer and tetracycline using isothermal titration calorimetry

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

metabolic profiling to determine bactericidal or bacteriostatic effects of new natural products using isothermal microcalorimetry

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe...

fluorescence anisotropy to determine transcription factor-dna binding affinity

This video describes high-performance fluorescence anisotropy that helps to monitor the interaction between transcription factors and DNA. The assay monitors the interactions of a fluorophore-labeled DNA molecule with its specific transcription factor by measuring the degree of polarization due to molecular rotation or anisotropy of the fluorophore-labeled...

Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

titration elisa as a method to determine the dissociation constant of receptor ligand interaction

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized...

analyzing dna-protein interactions with streptavidin-based biolayer interferometry

Author Spotlight: Utilizing Biolayer Interferometry to Analyze Protein-DNA Interactions &#8212; Insights into Genomic Stability Mechanisms

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the...

Quantitative Analysis of RPA-ssDNA Binding Kinetics Using Label-Free Methods

https://cloudfront.jove.com/CDNSource/teasers/66534_b.jpg

For each titration well, add 1.4 nanomoles reference DNA, transcription factor protein at a final concentration of 20 to 60 nanomoles, 0.2 millimolar DTT, and the binding buffer to 240 microliters of the melted...

the development and application of biophysical assays for evaluating ternary complex formation induced by proteolysis targeting chimeras (protacs)

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN).  Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

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differential scanning calorimetry to study dna aptamer-thermolabile ligand interaction

This video demonstrates differential scanning calorimetry &#8212; a technique to study the interaction between a biomolecule and a ligand. The binding of a DNA aptamer and a thermolabile ligand is studied by applying repeated heating and cooling cycles to the sample and obtaining scans for thermodynamic...

Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

using x-ray crystallography, biophysics, and functional assays to determine the mechanisms governing t-cell receptor recognition of cancer antigens

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

an elisa based binding and competition method to rapidly determine ligand-receptor interactions

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

accelerating rate calorimetry and complementary techniques to characterize battery safety hazards

Accelerating Rate Calorimetry and Complementary Techniques to Characterize Battery Safety Hazards

A method to characterize the potential failure hazards of lithium batteries is achieved with accelerating rate calorimetry. Heat and pressure release, visual observation of the failure event, and the capture of evolved gases are collected in this experiment to identify the worst credible threats of batteries taken to...

high sensitivity measurement of transcription factor-dna binding affinities by competitive titration using fluorescence microscopy

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery...

calculating the o2 concentration at the rpe monolayer to determine if cells are in hypoxic, normoxic, or hyperoxic conditions

Calculating the O2 Concentration at the RPE Monolayer to Determine if Cells are in Hypoxic, Normoxic, or Hyperoxic Conditions

the efficacy and underlying pathway mechanisms of shidugao treatment for anus eczema based on geo datasets and network pharmacology

Author Spotlight: Exploring ShiDuGao's Multi-Target Approach in Anus Eczema Treatment

This investigative effort sought to elucidate the mechanism of topical drug administration using a synergistic integration of network pharmacology and gene expression omnibus (GEO) datasets. This article evaluated the feasibility, target, and mechanism of ShiDuGao (SDG) in treating anus eczema.

The Efficacy and Underlying Pathway Mechanisms of ShiDuGao Treatment for Anus Eczema Based on GEO Datasets and Network Pharmacology

measuring biomolecular dsc profiles with thermolabile ligands to rapidly characterize folding and binding interactions

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning...

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 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 ...

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 ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

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 ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.