Name: the extraction of liver glycogen molecules for glycogen structure determination
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Description: Watch this Scientific Journal Video about The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination at JoVE.com

The authors are grateful to Mr. Gaosheng Wu and Miss Yunwen Zhu for technical assistance with FACE and Mr. Zhenxia Hu and Mr. Dengbin for technical assistance with SEC. MAS is supported by an Advance Queensland Industry Research Fellowship, Mater Foundation, Equity Trustees, and the L G McCallam Est and George Weaber Trusts. This work was supported by the Priority Academic Program of Jiangsu Higher Education Institutions, a Natural Science Foundation of China grant C1304013151101138, and the 2017 Jiangsu Innovation and Entrepreneurship talents program. Figure 1-5 were created using BioRender.

Mouse livers used to optimize this procedure21 were from 12 male BKS-DB/Nju background mice (7.2 weeks old, see the Table of Materials). Animal use was approved by Renmin Hospital of Wuhan University Animal Care and Ethics Committee, IACUC Issue No. WDRM 20181113.
1 Animal tissues
<ol>
	<li>Weigh mouse liver (1-1.8 g of whole liver from each mouse).</li>
	<li>Rapidly freeze the mouse liver in liquid nitrogen and store it at -80 &#176;C.</li>
</ol>
<p ...

<ol>
	<li>Hills, D. M., Heller, H. C., Hacker, S. D., Hall, D. W., Sadava, D., Laskowski, M., Freeeman, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Life: the science of biology. 12th edn. , (2020).</li><li>Calder, P. C., 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=Glycogen+of+high+molecular+weight+from+mammalian+muscle.">Glycogen of high molecular weight from mammalian muscle.</a> Carbohydrate Research. 135 (2), 249-256 (1985).</li><li>Rybicka, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosomes+-+The+organelles+of+glycogen+metabolism.">Glycosomes - The organelles of glycogen metabolism.</a> Tissue and Cell. 28 (3), 253-265 (1996).</li><li>Takeuchi, T., Iwamasa, T., Miyayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafine+structure+of+glycogen+macromolecules+in+mammalian-tissues.">Ultrafine structure of glycogen macromolecules in mammalian-tissues.</a> Journal of Electron Microscopy. 27 (1), 31-38 (1978).</li><li>Drochmans, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologie+du+glycogene+-+etude+au+microscope+electronique+de+colorations+negatives+du+glycogene+particulaire.">Morphologie du glycogene - etude au microscope electronique de colorations negatives du glycogene particulaire.</a> Journal of Ultrastructure Research. 6, 141-163 (1962).</li><li>Besford, Q. 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=The+structure+of+cardiac+glycogen+in+healthy+mice.">The structure of cardiac glycogen in healthy mice.</a> International Journal of Biological Macromolecules. 51 (5), 887-891 (2012).</li><li>Lumsden, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+structure+of+glycogen+in+some+cyclophyllidean+and+trypanorhynch+cestodes.">Macromolecular structure of glycogen in some cyclophyllidean and trypanorhynch cestodes.</a> Journal of Parasitology. 51 (4), 501-515 (1965).</li><li>Deng, B., 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=Molecular+structure+of+glycogen+in+diabetic+liver.">Molecular structure of glycogen in diabetic liver.</a> Glycoconjugate Journal. 32 (3-4), 113-118 (2015).</li><li>Deng, B., 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=The+mechanism+for+stopping+chain+and+total-molecule+growth+in+complex+branched+polymers,+exemplified+by+glycogen.">The mechanism for stopping chain and total-molecule growth in complex branched polymers, exemplified by glycogen.</a> Biomacromolecules. 16 (6), 1870-1872 (2015).</li><li>Jiang, X., 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=The+molecular-size+dependence+of+glycogen+enzymatic+degradation+rate+and+its+importance+for+diabetes.">The molecular-size dependence of glycogen enzymatic degradation rate and its importance for diabetes.</a> European Polymer Journal. 82 (1), 175-180 (2016).</li><li>Hu, Z., 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+structure+in+type+1+diabetic+mice:+towards+understanding+the+origin+of+diabetic+glycogen+molecular+fragility.">Glycogen structure in type 1 diabetic mice: towards understanding the origin of diabetic glycogen molecular fragility.</a> International Journal of Biological Macromolecules. 128, 665-672 (2019).</li><li>Suzuki, Y., 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=Insulin+control+of+glycogen+metabolism+in+knockout+mice+lacking+the+muscle-specific+protein+phosphatase+PP1G/R-GL.">Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/R-GL.</a> Molecular and Cellular Biology. 21 (8), 2683-2694 (2001).</li><li>Stetten, M. R., Katzen, H. M., Stetten, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+inhomogeneity+of+glycogen+as+a+function+of+molecular+weight.">Metabolic inhomogeneity of glycogen as a function of molecular weight.</a> Journal of Biological Chemistry. 122 (2), 587-599 (1956).</li><li>Nahorski, S. R., Rogers, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymic+fluorometric+micro+method+for+determination+of+glycogen.">An enzymic fluorometric micro method for determination of glycogen.</a> Analytical Biochemistry. 49 (2), 492-497 (1972).</li><li>Wang, Z., 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=Molecular+structural+features+of+glycogen+in+the+kidneys+of+diabetic+rats.">Molecular structural features of glycogen in the kidneys of diabetic rats.</a> Carbohydrate Polymers. 229, 115526 (2020).</li><li>Wang, L., 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=Molecular+structure+of+glycogen+in+Escherichia+coli.">Molecular structure of glycogen in Escherichia coli.</a> Biomacromolecules. 20 (7), 2821-2829 (2019).</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> Biochemical and Biophysical Research Communications. 362, 811-815 (2007).</li><li>Ryu, J. -. H., 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=Comparative+structural+analyses+of+purified+glycogen+particles+from+rat+liver,+human+skeletal+muscle+and+commercial+preparations.">Comparative structural analyses of purified glycogen particles from rat liver, human skeletal muscle and commercial preparations.</a> International Journal of Biological Macromolecules. 45 (5), 478-482 (2009).</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 (4), 1094-1100 (2010).</li><li>Tan, X., 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=A+new+non-degradative+method+to+purify+glycogen.">A new non-degradative method to purify glycogen.</a> Carbohydrate Polymers. 147 (1), 165-170 (2016).</li><li>Wang, Z., Liu, Q., Wang, L., Gilbert, R. G., Sullivan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+liver+glycogen+extraction+when+considering+molecular+fine+structure.">Optimization of liver glycogen extraction when considering molecular fine structure.</a> Carbohydrate Polymers. 261, 117887 (2020).</li><li>Zhao, Y., Tan, X., Wu, G., Gilbert, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+fine+structure+to+identify+optimal+methods+of+extracting+starch.">Using molecular fine structure to identify optimal methods of extracting starch.</a> Starch - Starke. 72, 1900214 (2020).</li><li>Shokri-Afra, H., Ostovar-Ravari, A., Rasouli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+classical+assay+method+for+liver+glycogen+fractions:+ASG+is+the+main+and+metabolic+active+fraction.">Improvement of the classical assay method for liver glycogen fractions: ASG is the main and metabolic active fraction.</a> European Review for Medical and Pharmacological Sciences. 20, 4328-4336 (2016).</li><li>Kerly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+solubility+of+glycogen.">The solubility of glycogen.</a> The Biochemical Journal. 24, 67-76 (1930).</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=Improving+size-exclusion+chromatography+for+glycogen.">Improving size-exclusion chromatography for glycogen.</a> Journal of Chromatography A. 1332 (1), 21-29 (2014).</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=Skeletal+muscle+glycogen+chain+length+correlates+with+insolubility+in+mouse+models+of+polyglucosan-associated+neurodegenerative+diseases.">Skeletal muscle glycogen chain length correlates with insolubility in mouse models of polyglucosan-associated neurodegenerative diseases.</a> Cell Reports. 27 (5), 1334-1344 (2019).</li><li>Orrell, S. A., Bueding, 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+comparison+of+products+obtained+by+various+procedures+used+for+the+extraction+of+glycogen.">A comparison of products obtained by various procedures used for the extraction of glycogen.</a> Journal of Biological Chemistry. 239 (12), 4021-4026 (1964).</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=Molecular+insights+into+glycogen+alpha-particle+formation.">Molecular insights into glycogen alpha-particle formation.</a> Biomacromolecules. 13 (11), 3805-3813 (2012).</li></ol>

Previous studies have shown that the structure of glycogen is important for its properties; for example, the molecular size affects the degradation rate of glycogen10, and the chain length distribution affects its solubility26. To properly understand these relationships, it is important to extract glycogen with a procedure that isolates, as much as possible, a representative and undamaged sample. Traditional methods of extraction utilized either hot alkaline conditions or c...

Glycogen is a complex, hyperbranched polymer of glucose found in animals, fungi, and bacteria1. In mammals, liver glycogen functions as a blood glucose buffer, preserving homeostasis, while muscle glycogen acts as a short-term glucose reservoir to provide energy directly2. The structure of glycogen is often described by three levels (shown in Figure 1): 1. Linear chains are formed by glucose monomers via (1&#8594;4)-&#945; glycosidic bonds, with branch points being connected via (1&#8594;6)-&#945; glycosidic bonds; 2. highly branched &#946; particles (~20 nm in diameter) that, especially in tissues such as skeletal muscle, act as independent glycogen molecules3,4; 3. larger &#945; glycogen particles (up to 300 nm in diameter) that consist of smaller &#946; glycogen units, which are found in the liver5, heart6, and in some non-mammalian species7. Hepatic &#945; particles from diabetic mice are molecularly fragile, with a propensity to degrade to &#946;-particles when dissolved in dimethyl sulfoxide (DMSO), while &#945; particles from non-diabetic controls generally remain unchanged. One hypothesis is that this fragility may exacerbate the poor blood glucose balance seen in diabetes, with the fragile &#945; particles potentially resulting in higher proportions of the more rapidly degraded &#946; particle8,9,10,11.
Traditional glycogen extraction methods utilize the relatively harsh conditions of exposing the liver tissue to hot alkaline solution12 or acid solutions such as trichloroacetic acid (TCA)13 or perchloric acid (PCA)14. While effective at separating the glycogen from other components of the liver tissue, these methods inevitably degrade the glycogen structure to some extent15,16. Although these methods are suitable for quantitative measurement of the glycogen content, they are not ideal for studies focused on obtaining structural information on the glycogen due to this structural damage. Since the development of these methods, a milder extraction procedure has been developed that utilizes cold Tris buffer (shown to inhibit glucosidase degradation) with sucrose density gradient ultracentrifugation17,18,19. With the pH controlled at ~8, this method does not subject the glycogen to the acid or alkaline hydrolysis seen in previous procedures.
Sucrose density gradient ultracentrifugation of homogenized liver tissue can separate glycogen particles from the majority of cell material. If necessary, additional purification can be performed by preparative size exclusion chromatography, resulting in the collection of purified glycogen with attached glycogen-associating proteins20. Although this method, with milder conditions, is more likely to preserve the structure of glycogen, it is difficult to prevent some portion of the glycogen from being lost in the supernatant, especially smaller glycogen particles that are less dense15. Another potential cause of glycogen loss is that the milder conditions allow some enzymatic degradation, resulting in lower glycogen yields compared to harsher extraction methods. Recent research reported optimization of the liver-glycogen extraction method to preserve the structure of glycogen21. Here, various sucrose concentrations (30%,50%, 72.5%) were tested to determine whether lower sucrose concentrations minimized the loss of smaller glycogen particles. The rationale was that the lower density would allow for smaller, less dense particles to penetrate the sucrose layer and aggregate in the pellet with the rest of the glycogen.
In this study, the extraction methods with and without a 10 min boiling step were compared to test whether glycogen degradation enzymes could be denatured, resulting in the extraction of more glycogen that was also free from partial degradation. Whole molecular size distributions and the glycogen chain length distributions were used to determine the structure of the extracted glycogen, similar to a starch extraction optimization published previously22. Size exclusion chromatography (SEC) with differential refractive index (DRI) detection was used to obtain the size distributions of glycogen, which describe the total molecular weight as a function of molecular size. Fluorophore-assisted carbohydrate electrophoresis (FACE) was used to analyze the chain-length distributions, which describe the relative number of glucoside chains of each given size (or degree of polymerization). This paper describes the methodology of extracting glycogen from liver tissues based on the previous optimization study21. The data suggest that the method most suited to preserve glycogen structure is a sucrose concentration of 30% with a 10 min boiling step.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-aminopyrene-1,3,6-trisulfonate (APTS)</td>
			<td>SIGMA Aldrich</td>
			<td>9341</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>SIGMA Aldrich</td>
			<td>695092</td>
			<td>0.1 M, pH 3.5 solution</td>
		</tr>
		<tr>
			<td>Agilent 1260 Infinity SEC system</td>
			<td>Agilent, Santa Clara, CA, USA</td>
			<td></td>
			<td>Size-exclusion chromatography (SEC)</td>
		</tr>
		<tr>
			<td>BKS-DB/Nju background mice</td>
			<td>Nanjing Biomedical Research Institution of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose Assay Kit (GOPOD Format)</td>
			<td>Megazyme</td>
			<td>K-GLUC</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenedinitrilotetraacetic acid (EDTA)</td>
			<td>SIGMA Aldrich</td>
			<td>431788</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>IKA</td>
			<td>T 25</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA Aldrich</td>
			<td>2104</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA Aldrich</td>
			<td>2104</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>P/ACE MDQ plus system</td>
			<td>Ab Sciex, US</td>
			<td></td>
			<td>Fluorophore-assisted carbohydrate electrophoresis (FACE)</td>
		</tr>
		<tr>
			<td>Refractive index detector</td>
			<td>Optilab UT-rEX, Wyatt, Santa Barbara, CA, USA)</td>
			<td></td>
			<td>Size-exclusion chromatography (SEC)</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>SIGMA Aldrich</td>
			<td>241245</td>
			<td>1 M, pH 4.5 solution</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>SIGMA Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SIGMA Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride</td>
			<td>SIGMA Aldrich</td>
			<td>156159</td>
			<td>1 M solution</td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>SIGMA Aldrich</td>
			<td>201154</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA Aldrich</td>
			<td>43617</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>Sodium nitrate</td>
			<td>SIGMA Aldrich</td>
			<td>NISTRM8569</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate</td>
			<td>SIGMA Aldrich</td>
			<td>221368</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>SIGMA Aldrich</td>
			<td>V90016</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPREMA pre-column, 1,000 and 10,000 columns</td>
			<td>Polymer Standards Services, Mainz, Germany</td>
			<td></td>
			<td>Size-exclusion chromatography (SEC)</td>
		</tr>
		<tr>
			<td>Trizma</td>
			<td>SIGMA Aldrich</td>
			<td>T 1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman</td>
			<td></td>
			<td>4 mL, Open-Top Thinwall Ultra-Clear Tube, 11 x 60 mm</td>
		</tr>
	</tbody>
</table>

the extraction of liver glycogen molecules for glycogen structure determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

While the procedure described above is for the most optimal method (30% sucrose with the addition of a 10min boiling step), data are provided here for glycogen extracted via three sucrose concentrations (30%, 50%, 72.5%), with and without a boiling step, as previously described21. Following the protocol, the purity, crude yield, and glycogen yield of dry glycogen extracted by different conditions are given in Table 1, reproduced from21. There were no signif...

Watch this Scientific Journal Video about The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination at JoVE.com

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

This protocol allows for the extraction of the liver glycogen molecules in a manner that preserves their structure and limits the amount of the small glycogen particles lost. With previous research showing diabetes alter glycogen structure, this method could provide in studying to diabetes and the various glycogen storage disease. To extract glycogen from the liver, start by transferring one gram frozen liver tissue to a 15 milliliter tube containing six milliliters of glycogen isolation buffer.While keeping on ice, homogenize the liver tissue using a tissue homogenizer. When done, transfer half or three milliliters of the cell suspension to a new tube, followed by boiling for 10 minutes. Keep the other half of the suspension on ice to extract glycogen-containing associated proteins that are not denatured.For the glycogen content determination, remove an eight microliter aliquot from each tube and keep the tube on ice. After spinning the remaining suspension at 6, 000 G for 10 minutes at four degrees Celsius, transfer the supernatant to ultracentrifuge tubes to centrifuge them at 3.6 times 10 to the fifth G and four degrees Celsius for 90 minutes. After discarding the remaining supernatants, resuspend the pellets in 1.5 milliliters of glycogen isolation buffer.Then, layer the samples over 1.5 milliliters of 30%sucrose solution in four milliliter ultracentrifuge tubes, followed by centrifugation as described before for two hours. After discarding the supernatants, resuspend the pellets in 200 microliters of deionized water. To precipitate glycogen from the cell suspension, add 800 microliters of absolute ethanol to the cell suspensions and mix well.Then, store the mixtures at minus 20 degrees Celsius for at least one hour to allow precipitation. Once precipitation occurs, centrifuge the samples at 6, 000 G for 10 minutes at four degrees Celsius and resuspend the glycogen pellet in 200 microliters of deionized water to repeat the ethanol precipitation process thrice. From the final cell suspension, remove an aliquot of eight microliters from each tube for glycogen content determination.Then, freeze the remaining supernatants in liquid nitrogen and freeze dry overnight. The next day, store the dried glycogen samples in the freezer at minus 20 degrees Celsius. The study analyzed the purity, crude yield, and glycogen yield of the dried glycogen sample extracted by different conditions.There were no significant differences in crude yield and glycogen yield between the groups extracted with the various conditions. In contrast, the glycogen purity was significantly influenced by the sucrose concentrations and the addition of a boiling step. Glycogen with the highest purity was extracted using the 30%sucrose concentration and a 10-minute boiling step.Glycogen was extracted from the liver homogenate by either 30%50%or 72.5%sucrose, boiled or unboiled, to assess the effects of the various conditions on the size of the molecules in the final extract. Chain length analysis was performed on six livers for both boiled and unboiled, using 30%50%and 72.5%sucrose concentrations. The content of the glycogen molecules or beta particles with a hydrodynamic radius, RH, lower than 30 nanometers was calculated.The boiled samples had lower average RH values and a higher particle content than the unboiled samples. Also, lower sucrose concentrations resulted in lower RH values and higher beta particle contents. The introduction of a boiling step also led to lower RH max or the maxima values, while the sucrose concentration had no significant effect.Make sure tissue is fully homogenized and there are no chunks of the tissue remaining. So, this techniques paves the way for us to explore what paths links the smaller beta particles together to form the larger alpha particles.

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Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...