We are grateful to the Dr. Thierry Pourcher for allowing us to use the fluorescence spectrometer and for his help. The laboratory is funded by the Ligue Nationale Contre le Cancer (&#233;quipe labellis&#233;e), the Association pour la Recherche contre le Cancer, the Institut National du Cancer (INCa), the Agence Nationale pour la Recherche, METOXIA (EU program FP7), the Centre A. Lacassagne, the Centre National de la Recherche Scientifique, the Institut National de la Sant&#233; et de la Recherche M&#233;dicale and the University of Nice (<a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a>). We thank Dr. M Christiane Brahimi-Horn for critical reading and editorial correction.

Biochemical Titration of Glycogen
1. Cell Lysis
<ol>
	<li>Seed cells at a concentration of 0.5-2 x 106 per 100 mm diameter dish.</li>
	<li>Treatment: incubate cells 24, 48, or 72 hr in low oxygen concentration (hypoxia) in a Bug-Box anaerobic work station (Ruskinn Technology Biotrace International Plc, Bridgend, UK) set at 1% or 0.1% O2, 94% or 94.9% N2, and 5% CO2. In parallel, incubate cells in normoxia (21% O2, 5% CO2). Change medium (25 mM glucose-containing medium) every 24 hr to minimize variations in the glucose concentration during the time of experiment.</li>
	<li>After treatment, wash cells 2x with PBS to remove traces of glucose from culture medium. Scrape the cells in cold PBS.</li>
	<li>Centrifuge the solution at 1,000 rpm for 5 min, discard the supernatant and wash the pellet once with PBS. The pellet can be frozen at -20 &#176;C before proceeding further.</li>
	<li>Resuspend the pellet in 100 &#956;l of distilled water. Add 100 &#956;l of the Glycogen Hydrolysis Buffer provided with the Glycogen Assay Kit (Abcam). Boil the lysate at 95 &#176;C for 5 min to inactivate enzymes.</li>
	<li>Vortex and centrifuge the lysate at 13,000 rpm for 10 min to remove the insoluble products. Keep the supernatant.</li>
	<li>To normalize glycogen levels to the protein content between samples, quantification of proteins can be done with 25-35 &#956;l of the supernatant using the Bicinchoninic Acid assay (BCA-Interchim).</li>
</ol>
Some of the lysate can be used to measure the free glucose concentration inside the cells.
2. Hydrolysis of Glycogen
<ol>
	<li>Mix 50 &#956;l of the supernatant (step 1) with 1 &#956;l of the Hydrolysis Enzyme Mix. This mixture contains glucoamylase. Incubate for 30 min at room temperature.</li>
	<li>Prepare the calibration curve by diluting the standard glycogen (2 mg/ml) supplied with the kit to a solution of 10 &#956;g/ml in the hydrolysis buffer. Next, prepare six separate tubes with 0, 4, 8, 12, 16, and 20 &#956;l of 10 &#956;g/ml standard and adjust each tube to a final volume of 50 &#956;l with hydrolysis buffer to give a concentration of glycogen of 0, 0.04, 0.08, 0.12, 0.16, and 0.2 &#956;g/ml. Add 1 &#956;l of Hydrolysis Enzyme Mix in the standards and incubate for 30 min at room temperature.</li>
</ol>
The background glucose concentration of the cell line, given by value for the free glucose concentration, must be subtracted from the value for the total glucose concentration (glucose from the glycogen hydrolysis + free-glucose) to determine the level of glycogen in the cell.
3. Development and Reading of Fluorescence
<ol>
	<li>For each condition, prepare one tube (tube 1) to measure free glucose concentration and two tubes (tubes 2 and 3) to measure total glucose concentration (each tube with a different volume of hydrolyzed glycogen).</li>
	<li>Add 15 &#956;l of supernatant extracted at the end of step 1&#160;in tube 1. Add 35 &#956;l of hydrolysis buffer to complete to a final volume of 50 &#956;l. Obtained fluorescence will give the free glucose concentration.</li>
	<li>Add X &#956;l of hydrolyzed glycogen (obtained in step 2) in tube 2. Adjust to a final volume of 50 &#956;l. Sample volume (X &#956;l) has to be adjusted according to cell density and/or cell type in order to obtain fluorescence values that are not beyond concentrations of the calibration curve.</li>
	<li>Add 2X &#956;l of hydrolyzed glycogen in tube 3. Adjust to a final volume of 50 &#956;l.</li>
	<li>Prepare a development solution for samples and standards by mixing for each tube 48.7 &#956;l of Development Buffer, 1 &#956;l of Development Enzyme Mix and 0.3 &#956;l of OxiRed Probe (supplied in the Glycogen Assay Kit). For example, for 2 conditions, prepare a mix for 12 tubes (6 for standards, 2 for free glucose and 4 for total glucose fluorescence).</li>
	<li>Add 50 &#956;l of this mix to each tube and incubate for 30 min at room temperature in the dark.</li>
	<li>Measure the fluorescence&#160;at an excitation wavelength of 535 nm, an emission wavelength of 587 nm as recommended, and a slit of 3 nm for excitation and emission.</li>
</ol>

<ol>
	<li>Pelletier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. 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+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

The authors declare no conflict of interest.

Biochemical titration of glycogen in vitro allows an accurate quantification of cells glycogen content. Comparing to some other techniques (PAS, immunofluorescence with a glycogen antibody, etc.), this titration is very specific, sensitive and reproducible. Moreover, the method is convenient since it requires no radioactivity but a fluorescence spectrometer. However, this technique is purely quantitative and does not provide information about glycogen distribution in the cell.
<p class="jove_content...

Glycogen is a multibranched polymer of glucose residues, which is present in the cytoplasm of many cell types. It is one of the main forms of energy storage in cells and plays an important role in glucose metabolism. Most mammalian cells are able to produce and store glycogen, which can be rapidly degraded into glucose to promote glycolysis and ATP production during metabolic stress. Hepatocytes produce massive amounts of glycogen to regulate the blood sugar level thereby providing a continuous supply of glucose to the body. In contrast, the concentration of glycogen in other cells (muscles, red blood cells,&#160;etc.) is relatively low. However, locally, these quantities are sufficient to provide energy in the&#160;short-term when the cells are suddenly exposed to an environment deprived of nutrients.
Glycogen synthesis and glycogen breakdown follows the same steps in all tissues (Figure 1). Firstly, glucose enters cells by glucose transporters (GLUTs), and is rapidly converted from glucose-6-phosphate (G6P) into glucose-1-phosphate (G1P) by phosphoglucomutase. G1P is then modified into UDP-glucose, and the carbon C1 of UDP-glucose is attached to a tyrosine residue of glycogenin, the anchoring protein of glycogen. This molecule, considered a glycogen primer, is extended by attachment of the UDP-glucose to the terminal glucose by an &#945;(1&#8594;4) bond via the glycogen synthase. Finally, when a linear chain of over 11 glucose residues is formed, the branching enzyme transfers a terminal oligosaccharide constituted by a minimum of 6 glucose residues to another glucose of the chain nearby through an &#945;(1&#8594;6) linkage. The repetition of this process gives a massive fractal structure containing branches that form a helix with 6.5 glucose per turn. Glycogen can be reversely hydrolyzed to glucose by the concerted action of debranching enzymes that hydrolyze the &#945;(1&#8594;6) bound and glycogen phosphorylase that hydrolyzes the &#945;(1&#8594;4) glycosidic bound between the last residue of glucose of a branch and the glycogen molecule. This reaction called glycogenolysis is activated by increasing levels of AMP (reflecting ATP consumption), and inhibited by glucose and ATP2,3.
By electron microscopy, glycogen molecules have been described in many cell types as &#946; free particles (or glycogen monoparticles) of 15-30 nm in diameter. In specific cell types such as hepatocytes, &#946; particles can be assembled into a complex to form rosettes, also known as &#945; particles that vary in diameter from 80 nm to a maximum of 200 nm4. The way these &#946; particles are bonded to form larger clusters of &#945; particles is still not fully elucidated. Some evidence tends to prove that &#946; particles can be bonded by covalent bonding5, hydrogen bonding, or even through protein-protein interactions6. The amount of glycogen stored in the cells depends on many parameters: (I) the amount of glycogenin in the cell that initiates glycogen synthesis; (II) the activity of glycogen synthase and phosphorylase regulated by protein phosphorylation/dephosphorylation; (III) the concentration of glucose in the cells, which is dependent on several parameters such as the supply of glucose from the vascular system and glucose uptake by cells. Glycogen stores are tightly regulated by allosteric regulation of biosynthetic hormones through intermediate metabolites, by hormones regulating energy metabolism, and by nutrient sensing signaling pathways7.
It is important to be able to quantify glycogen in biological samples to better understand the importance of glycogen metabolism at the whole body and cellular level. We describe here a precise, reproducible and convenient biochemical in vitro assay for glycogen. This technique is based on the quantification glucose fluorescence before and after specific hydrolysis of glycogen.
Other methods exist to estimate the level of glycogen in the cells, but most of them are not quantitative. One of the first techniques described for quantification of glycogen in cells was based on measurement of [14C]-glucose incorporation into glycogen8,9. The use of radioactivity makes this process more difficult to handle but it has the advantage of providing the rate of glucose incorporation into glycogen and in distinguishing between the distribution of glucose residues on the outer branches and in the core of the molecule (it also requires an additional &#946;-amylolysis and a chromatographic step). Another technique was developed more recently and is based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino}-2-deoxyglucose), a 2-deoxyglucose fluorescent derivative, into glycogen10. The measured fluorescence intensity reflects the amount of glycogen produced and can be measured with a fluorescence reader. Distribution of fluorescence in the cell can also be evaluated by confocal microscopy.
Among the other nonquantitative techniques, Periodic Acid-Schiff staining (PAS) is perhaps the most common. It can be used for the detection of glycogen in fixed cells, tissue sections in paraffin or frozen. This histological technique colors non specifically polysaccharides, glycolipids, glycoproteins, cellulose and neutral mucins in purple. The specificity of this test can be increased by treatment of fixed cells or tissue sections with diastase, which specifically digests glycogen. Thereafter, the level of glycogen can be qualitatively estimated by comparing unhydrolyzed samples (all carbohydrate modified macromolecules) to hydrolyzed samples (carbohydrate modified macromolecules except glycogen). PAS staining and microscopic analysis, unlike biochemical assays of glycogen, provides information concerning the distribution of glycogen in the cell, which can be diffused or concentrated in a certain part of the cell. However, even though PAS staining estimates differences in glycogen accumulation between different conditions, it is not quantitative11.
A monoclonal mouse antibody originally made using mandibular condylar cartilage as the antigen has been shown to react with glycogen in cells and with purified glycogen in vitro12. As this antibody specifically recognizes glycogen-related sugar chains, it is a useful tool for the detection of glycogen by immunohistochemistry in a more specific way than the PAS staining.
Electron microscopy is another technique that allows visualization of grains of glycogen in cells and evaluation of the degree of glycogen storage. In fact, glycogen &#946; particles are easily recognizable with an electron microscopy as electron dense granules1.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
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			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
	 </tr>
		 <tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
			<td></td>
		 </tr>
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 </table>

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.

A low level of oxygen (hypoxia) in tumors signals to tumor cells the need to store energy to handle subsequent nutrient depletion, so as to survive. As glycogen is the main energetic polymer of glucose in mammalian cells, we studied the regulation of glycogen storage in hypoxia. The calculation and standardization of the concentration of glycogen in cell lysates must be performed on the raw data of fluorescence as shown in Tables 1, 2, and 3. The biochemical assay for gl...

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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.

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 ...

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28.2K Views.  University of Nice - Sophia Antipolis. 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.

Video: Biochemical Titration of Glycogen In vitro

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 ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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 ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...