The authors acknowledge Nordic Energy Research (AquaFEED, project no. 24), Innovationfonden Denmark (Pant Power, project no. 12-131844), and Villum Fonden (project no. 13363)

1. Preparation
<ol>
	<li>Cyanobacterial cultures
	<ol>
		<li>Grow Synechocystis sp. PCC 6803 at 30 &#176;C in liquid BG11 medium8, with a constant supply of air supplemented with 1% (v/v) CO2. Illuminate the cultures continuously with light at a photosynthetic photon flux density of 50 &#181;mol photon/m2/s.</li>
		<li>Grow Synechococcus sp. PCC 7002 in liquid A+ medium23 (BG11 medium can also be used), with a constant supply of air supplemented with 1% (v/v) CO2. The temperature should be 37 &#176;C. Illuminate the cultures continuously with light at a photosynthetic photon flux density of 150 &#181;mol photon/m2/s.</li>
		<li>Measure the optical density (OD) of the culture at 730 nm using a cuvette with a light path of 1 cm. If the OD value is above 0.8, make appropriate dilutions to obtain OD measurements that are proportional to the cell concentration. 
		NOTE: The protocols presented below are suitable for liquid cultures, with a cell density corresponding to an OD730nm value of 2 or higher. When cultures in the exponential growth phase are desired, which typically have an OD730nm value below 1, concentrate the cell density by centrifugation and resuspension in a buffer or medium to achieve an OD730nm value of 2 or higher.</li>
	</ol>
	</li>
	<li>Buffers and reagents
	<ol>
		<li>Make 50 mM Tris-HCl buffer at pH 8.</li>
		<li>Make 50 mM sodium acetate buffer at pH 5.</li>
		<li>Make a stock solution of 8 U/mL amyloglucosidase in 50 mM sodium acetate buffer, pH 5.</li>
		<li>Make a stock solution of 2 U/mL &#945;-amylase in 50 mM sodium acetate buffer, pH 5.</li>
		<li>Using distilled water, makeglucose standard solutions at concentrations ranging between 0 and 100 &#181;g/mL,.</li>
		<li>Prepare GOD-POD reagent from the D-Glucose Assay Kit (GODPOD Format), following the manufacturer&#180;s instruction.</li>
	</ol>
	</li>
</ol>
2. Determination of the Cell Dry Weight (Optional)
<ol>
	<li>Transfer 2 mL of a culture or a cell resuspension (see step 1.1) to a 2.0 mL tube and centrifuge at 6,000 x g and 4 &#176;C for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in 1 mL of water and centrifuge at 6,000 x g and 4 &#176;C for 5 min. Discard the supernatant and resuspend the cell pellet in 0.5 mL of water.</li>
	<li>Transfer the suspension to a pre-weighed aluminum tray. Transfer the tray to a drying oven at 105 &#176;C for overnight drying (approximately 18 h). 
	CRITICAL: It is important that the tray is handled with forceps to avoid the transfer of material from the fingers. Dry an empty, pre-weighed aluminum tray under the same conditions to determine any weight loss from the trays during drying.</li>
	<li>After drying, remove the tray from the oven and allow it to equilibrate at ambient conditions for 5 min before weighing it with an accuracy of 0.0001 g. 
	NOTE: The value can be used to normalize the glycogen content on a cell-dry-weight basis.</li>
</ol>
3. Lysis of Cyanobacterial Cells
<ol>
	<li>Transfer 1 mL of a culture or a cell resuspension (see step 1.1) to a 1.5 mL tube and centrifuge at 6,000 x g and 4 &#176;C for 10 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in 1 mL of 50 mM Tris-HCl, pH 8, and centrifuge at 6,000 x g and 4 &#176;C for 10 min. Discard the supernatant and resuspend the cell pellet in 1 mL of the Tris-HCl buffer. Repeat the process.</li>
	<li>Thoroughly resuspend the pellet in 500 &#181;L of 50 mM Tris-HCl buffer, pH 8. 
	CRITICAL: Resuspend the pellet well for an efficient cell lysis. Keep the resuspension in ice.</li>
	<li>Lyse the resuspended cells at 4 &#176;C by performing 30 cycles of ultrasonication, each cycle consisting of 30 s at a frequency of 20 kHz with the maximum amplitude, followed by 90 s without. 
	NOTE: This method can efficiently lyse Synechococcus sp. PCC 7002.
	<ol>
		<li>Alternatively, transfer the cell resuspension to a screw-cap tube and add zirconium oxide beads to the tube, following manufacturer&#39;s instructions. Set the tube in a tissue homogenizer and lyse the cells at 4 &#176;C by performing 2 cycles of beating, each cycle consisting of 5 min at the frequency setting of 5. 
		NOTE: This method can efficiently lyse Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002.</li>
	</ol>
	</li>
	<li>Centrifuge the tube containing the lysate for 10 min at 6,000 x g and 4 &#176;C. 
	NOTE: The pellet should mainly consist of large cell debris. If there is a significant number of unbroken cells, repeat step 3.4. Keep the supernatant on ice.</li>
	<li>Determine the protein concentration using a commercial BCA Protein assay kit. 
	NOTE: The value can be used to normalize the glycogen content on a protein content basis.</li>
</ol>
4. Glycogen Precipitation
<ol>
	<li>Remove chlorophyll a from the cell lysate by mixing 900 &#181;L of 96% (v/v) ethanol and 100 &#181;L of the supernatant from step 3.5 in a 1.5 mL screw-cap tube. After closing the cap, heat the tube at 90 &#176;C for 10 min using a standard laboratory heating block.</li>
	<li>Incubate the tube on ice for 30 min.</li>
	<li>Centrifuge the tube at 20,000 x g and 4 &#176;C for 30 min and carefully remove the supernatant; the pellet contains glycogen. Lightly dry the pellet in air to remove excess ethanol. 
	CRITICAL:Excessive drying of the pellet must be avoided, otherwise it becomes difficult to solubilize in step 5.1.</li>
	<li>OPTIONAL: Measure the absorbance of the supernatant obtained in step 4.3 at 664 nm to determine the chlorophyll a content. Use an absorption coefficient of 84.6 L/g/cm24. 
	NOTE: The value can be used to normalize the glycogen content.</li>
</ol>
5. Enzymatic Hydrolysis and Glycogen Determination
<ol>
	<li>Solubilize the pellet obtained in step 4.3 in 100 &#181;L of 50 mM sodium acetate, pH 5, and add 50 &#181;L of 8 U/mL amyloglucosidase and 50 &#181;L of 2 U/mL &#945;-amylase. Mix these materials well using a vortex. 
	NOTE: Mixing by pipetting is not recommended because the glycogen pellet is viscous.</li>
	<li>Incubate the mixture at 60 &#176;C on a heating block for 2 h to enable the digestion of glycogen into glucose molecules.</li>
	<li>Centrifuge the sample at 10,000 x g for 5 min and transfer the supernatant to a new 1.5 mL tube.</li>
</ol>
6. Determination of the Total Glucose Content Using the GOD-POD Reagent
<ol>
	<li>Measure the concentration of glucose in the supernatant obtained in step 5.3 using the GOD-POD reagent. Transfer 100 &#181;L of the supernatant from step 5.3 to a well in a 96-well plate. As a negative control, use 100 &#181;L of 50 mM sodium acetate, pH 5. For generating a calibration curve, also measure the glucose standard solutions.</li>
	<li>Add 150 &#181;L of GOD-POD reagent to each sample and quickly mix by pipetting.</li>
	<li>Incubate the plate statically at 25 &#176;C for 30 min. Record the absorbance value at 510 nm using a plate reader.</li>
	<li>Calculate the concentration of glucose using a calibration curve obtained from the glucose standards. 
	NOTE: The glycogen concentration in the cell lysate is expressed as the concentration of glucose (&#181;g/mL).</li>
</ol>

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The authors have nothing to disclose.

Critical steps within the protocol are glycogen precipitation and resuspension. After centrifugation following ethanol precipitation, glycogen forms a translucent pellet that loosely adheres to the walls of the microcentrifuge tubes. Therefore, when removing the supernatant, special attention needs to be given so as not to remove the pellet. The glycogen pellet is sticky, and solubilization can be difficult if it dries out. Note that the complete solubilization of the glycogen pellet is important because incomplete solub...

Cyanobacteria accumulate glycogen as the major carbohydrate store of carbon from CO2 fixed in light through photosynthesis. Glycogen is a glycan consisting of linear &#945;-1,4-linked glucan with branches created by &#945;-1,6-linked glucosyl linkages. Glycogen biosynthesis in cyanobacteria starts with the conversion of glucose-6-phosphate into ADP-glucose through the sequential action of phosphoglucomutase and ADP-glucose pyrophosphorylase. The glucose moiety in ADP-glucose is transferred to the non-reducing end of the &#945;-1,4-glucan backbone of glycogen by one or more glycogen synthases (GlgA). Subsequently, a branching enzymes introduce the &#945;-1,6-linked glucosyl linkage, which is further extended to generate the glycogen particle. In the dark, glycogen is broken down by glycogen phosphorylase, glycogen debranching enzymes, &#945;-glucanotransferase, and malto-dextrin phosphorylase into phosphorylated glucose and free glucose. These feed into catabolic pathways, including the oxidative pentose phosphate pathway, the Embden-Meyerhof-Parnas pathway (glycolysis), and the Entner-Doudoroff pathway1,2,3,4.
Glycogen metabolism in cyanobacteria has garnered increasing interest in recent years because of the potential for cyanobacteria to develop into microbial cell factories driven by sunlight to produce chemicals and fuels. Glycogen metabolism could be modified to increase the yield of the products, because glycogen is the largest flexible carbon pool in these bacteria. An example is the cyanobacterium Synechococcus sp. PCC 7002, which has been genetically engineered to produce mannitol; the genetic disruption of glycogen synthesis increases the mannitol yield 3-fold5. Another example is the production of bioethanol from glycogen-loaded wildtype Synechococcus sp. PCC 70026. The wildtype cell glycogen content may be up to 60% of the dry weight of the cell during nitrogen starvation6.
Our understanding of glycogen metabolism and regulation has also expanded in recent years. While glycogen is known to accumulate in the light and to be catabolized in the dark, detailed kinetics of glycogen metabolism during the diel cycle was only recently revealed in Synechocystis sp. PCC 68037. Moreover, several genes affecting the accumulation of glycogen have been identified. A notable example is the discovery that the putative histidine kinase PmgA and the non-coding RNA PmgR1 form a regulatory cascade and control the accumulation of glycogen. Interestingly, the pmgA and pmgR1 deletion mutants accumulate twice as much glycogen as the wildtype strain of Synechocystis sp. PCC 68038,9. Other regulatory elements are also known to affect the accumulation of glycogen, including the alternative sigma factor E and the transcriptional factor CyAbrB210,11.
As interest in glycogen regulation and metabolism grow, a detailed protocol describing the determination of the glycogen content is warranted. Several methods are used in the literature. Acid hydrolysis followed by the determination of the monosaccharide content through high-pressure anion exchange liquid chromatography coupled with a pulsed amperometric detector or spectrometric determination following treatments with acid and phenol are widely used methods to approximate the glycogen content9,10,12,13. However, a high-pressure anion exchange liquid chromatographic instrument is very expensive and does not discriminate glucose derived from glycogen from that derived from other glucose-containing glycoconjugates, such as sucrose14, glucosylglycerol15, and cellulose16,17,18, which are known to accumulate in some cyanobacterial species. The acid-phenol method can be performed using standard laboratory equipment. However, it uses highly toxic reagents and does not distinguish glucose derived from different glycoconjugates, nor does it distinguish glucose from other monosaccharides that constitute cellular materials, such as glycolipids, lipopolysaccharides, and extracellular matrices12. Notably, the hot acid-phenol assay is often used for the determination of total carbohydrate content rather than for the specific determination of glucose content12. Enzymatic hydrolysis of glycogen to glucose by &#945;-amyloglucosidase followed by the detection of glucose through an enzyme-coupled assay generates a colorimetric readout that is highly sensitive and specific to glucose derived from glycogen. The specificity can be enhanced further with the preferential precipitation of glycogen from cell lysates by ethanol5,8,19.
Here, we describe a detailed protocol for an enzyme-based assay of the glycogen content in two of the most widely studied cyanobacterial species, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, in the wildtype and mutant strains. In order to ensure efficient hydrolysis, a cocktail of &#945;-amylase and &#945;-amyloglucosidase is used8. The endo-acting &#945;-amylase hydrolyzes the &#945;-1,4-linkages in various glucans into dextrins, which are further hydrolyzed to glucose by exo-acting &#945;-amyloglucosidase20. The synergistic effects of these enzymes are well known, and these enzymes are routinely used for the selective hydrolysis of starch, which is an &#945;-linked glucan like glycogen, without affecting other glycoconjugants, such as cellulose, in the plant biomass21. The released glucose is quantitatively detected following an enzyme-coupled assay consisting of glucose oxidase-which catalyzes the reduction of oxygen to hydrogen peroxide and the oxidation of glucose to a lactone-and peroxidase-which produces a pink-colored quinoneimine dye from hydrogen peroxide, a phenolic compound, and 4-aminoantipyrine22.

<table><tbody><tr><td>QSonica Sonicators Q700</td><td>Qsonica, LLC</td><td>NA</td><td>QSonica</td></tr><tr><td>SpectraMax 190 Microplate Reader&amp;nbsp;</td><td>Molecular Devices</td><td>NA</td><td>Eliza plate reader</td></tr><tr><td>Bullet Blender Storm</td><td>Next Advance</td><td>BBY24M-CE</td><td>Beads beater</td></tr><tr><td>Ultrospec 3100 pro UV/Visible Spectrophotometer</td><td>Amersham Biosciences</td><td>NA</td><td>Spectrophotometer</td></tr><tr><td>Tris&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1503</td><td>Buffer</td></tr><tr><td>HCl</td><td>Merck</td><td>1-00317</td><td>pH adjutment</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>32319</td><td>Buffer</td></tr><tr><td>Amyloglycosidase (Rhizopus sp.)</td><td>Megazyme</td><td>E-AMGPU</td><td>Enzyme for glycogen depolymerization</td></tr><tr><td>&amp;alpha;-Amylase, thermostable (Bacillus licheniformis)</td><td>Sigma-Aldrich</td><td>A3176</td><td>Enzyme for glycogen depolymerization</td></tr><tr><td>D-Glucose</td><td>Merch</td><td>8337</td><td>Standard for the glucose assay</td></tr><tr><td>Pierce BCA Protein assay kit&amp;nbsp;</td><td>Thermo Fisher scientific</td><td>23225</td><td>For determination of protein concentrations</td></tr><tr><td>Aluminum drying trays, disposable</td><td>VWR</td><td>611-1362</td><td>For determination of cell dry weights</td></tr><tr><td>D-Glucose assay kit (GODPOD format)</td><td>Megazyme</td><td>K-GLUC</td><td>For determination of glucose concentrations</td></tr><tr><td>Zirconium oxide breads, 0.15 mm</td><td>Next Advance</td><td>ZrOB015</td><td>Beads for cell lysis in a Bullet Blendar Storm</td></tr><tr><td>RINO tubes</td><td>Next Advance</td><td>NA</td><td>Tubes for cell lysis in a Bullet Blendar Storm</td></tr></tbody></table>

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.

10 mL of wildtype Synechocystis sp. PCC 6803 were grown under photoautotrophic conditions until the OD730nm value reached approximately 0.8. The cells were harvested and resuspended in 50 mM Tris-HCl, pH 8. The OD730nm value was adjusted to 2-3. The glycogen content was analyzed following the protocol described above. The glycogen content per the OD730nm was 13 &#177; 1.8 &#181;g/mL/OD730nm (N = 12). The glycogen content relati...

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Determination of the Glycogen Content in Cyanobacteria

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 ...

determination-of-the-glycogen-content-in-cyanobacteria

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

12.9K Views.  Carlsberg Research Laboratory. 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.

Video: Determination of the Glycogen Content in Cyanobacteria

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Immunology and Infection

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 ...

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.

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 ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached to each other by &#945;-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, &#946;-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis FACE Method

In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen.

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached ...

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 ...

Biology

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

Source: Wilson, W. A., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
In this video, we describe a qualitative spectrophotometric assay to determine the extent of branching in a glycogen sample with an uncharacterized structure. Comparing the absorbance maxima of the uncharacterized sample with that of polysaccharides with known branched structures indicates the presence of branching in the uncharacterized sample.

Source: Wilson, W. A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
In this video, we describe a ...

Determination of the Glycogen Content in Cyanobacteria

Summary

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Chapters in this video

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Biochemical Titration of Glycogen In vitro

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

Glycogen Branching Assay to Measure the Degree of Glycogen Branching