Name: detection of glycosaminoglycans by polyacrylamide gel electrophoresis and silver staining
Uploaded: 2021-02-25T15:00:00
Description: Watch this Scientific Journal Video about Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining at JoVE.com

This work was funded by F31 HL143873-01 (WBL), R01 HL125371 (RJL and EPS)

All biological samples analyzed in this protocol were obtained from mice, under protocols approved by the University of Colorado Institutional Animal Care and Use Committee.
1. Heparan sulfate isolation
<ol>
	<li>Delipidation of tissue samples 
	NOTE: Delipidation is an optional step for fat-rich tissues.
	<ol>
		<li>Make a 1:1 mixture of methanol and dichloromethane. Prepare approximately 500 &#956;L per sample; larger pieces of tissue may require up to 1 mL.</li>
		<l...

<ol>
	<li>LaRivi&#232;re, W. B., Schmidt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pulmonary+endothelial+glycocalyx+in+ARDS:+A+critical+role+for+heparan+sulfate.">The pulmonary endothelial glycocalyx in ARDS: A critical role for heparan sulfate.</a> Current Topics in Membrane. 82, 33-52 (2018).</li><li>Haeger, S. M., Yang, Y., Schmidt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+in+the+developing,+healthy,+and+injured+lung.">Heparan sulfate in the developing, healthy, and injured lung.</a> American Journal of Respiratory Cell and Molecular Biology. 55 (1), 5-11 (2016).</li><li>Morita, H., Yoshimura, A., Kimata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+heparan+sulfate+in+the+glomerular+basement+membrane.">The role of heparan sulfate in the glomerular basement membrane.</a> Kidney International. 73 (3), 247-248 (2008).</li><li>Schmidt, E. P., 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+pulmonary+endothelial+glycocalyx+regulates+neutrophil+adhesion+and+lung+injury+during+experimental+sepsis.">The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.</a> Nature Medicine. 18 (8), 1217-1223 (2012).</li><li>Haeger, S. M., 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=Epithelial+heparan+sulfate+contributes+to+alveolar+barrier+function+and+is+shed+during+lung+injury.">Epithelial heparan sulfate contributes to alveolar barrier function and is shed during lung injury.</a> American Journal of Respiratry Cell and Molecular Biology. 59 (3), 363-374 (2018).</li><li>Mankin, H. J., Lippiello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glycosaminoglycans+of+normal+and+arthritic+cartilage.">The glycosaminoglycans of normal and arthritic cartilage.</a> Journal of Clinical Investigation. 50 (8), 1712-1719 (1971).</li><li>Annaval, T., 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=Heparan+sulfate+proteoglycans+biosynthesis+and+post+synthesis+mechanisms+combine+few+enzymes+and+few+core+proteins+to+generate+extensive+structural+and+functional+diversity.">Heparan sulfate proteoglycans biosynthesis and post synthesis mechanisms combine few enzymes and few core proteins to generate extensive structural and functional diversity.</a> Molecules. 25 (18), (2020).</li><li>Zhang, F., 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=Comparison+of+the+interactions+of+different+growth+factors+and+glycosaminoglycans.">Comparison of the interactions of different growth factors and glycosaminoglycans.</a> Molecules. 24 (18), (2019).</li><li>Pempe, E. H., Xu, Y., Gopalakrishnan, S., Liu, J., Harris, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+structural+selectivity+of+synthetic+heparin+binding+to+Stabilin+protein+receptors.">Probing structural selectivity of synthetic heparin binding to Stabilin protein receptors.</a> Journal of Biological Chemistry. 287 (25), 20774-20783 (2012).</li><li>Cowman, M. K., 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=Polyacrylamide-gel+electrophoresis+and+Alcian+Blue+staining+of+sulphated+glycosaminoglycan+oligosaccharides.">Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.</a> Biochemical Journal. 221 (3), 707-716 (1984).</li><li>M&#248;ller, H. J., Poulsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+silver+staining+of+glycoproteins+in+thin+sodium+dodecyl+sulfate+polyacrylamide+gels.">Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels.</a> Analytical Biochemistry. 226 (2), 371-374 (1995).</li><li>Min, H., Cowman, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+alcian+blue+and+silver+staining+of+glycosaminoglycans+in+polyacrylamide+gels:+Application+to+electrophoretic+analysis+of+molecular+weight+distribution.">Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: Application to electrophoretic analysis of molecular weight distribution.</a> Analytical Biochemistry. 155 (2), 275-285 (1986).</li><li>Jay, G. D., Culp, D. J., Jahnke, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+extensively+glycosylated+proteins+on+sodium+dodecyl+sulfate-polyacrylamide+gels:+Enhancement+by+carbohydrate-binding+dyes.">Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: Enhancement by carbohydrate-binding dyes.</a> Analytical Biochemistry. 185 (2), 324-330 (1990).</li><li>Abraham, E., 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=Liposomal+prostaglandin+E1+(TLC+C-53)+in+acute+respiratory+distress+syndrome:+a+controlled,+randomized,+double-blind,+multicenter+clinical+trial.+TLC+C-53+ARDS+Study+Group.">Liposomal prostaglandin E1 (TLC C-53) in acute respiratory distress syndrome: a controlled, randomized, double-blind, multicenter clinical trial. TLC C-53 ARDS Study Group.</a> Critical Care Medicine. 27 (8), 1478-1485 (1999).</li><li>Pervin, A., al-Hakim, A., Linhardt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+glycosaminoglycan-derived+oligosaccharides+by+capillary+electrophoresis+using+reverse+polarity.">Separation of glycosaminoglycan-derived oligosaccharides by capillary electrophoresis using reverse polarity.</a> Analytical Biochemistry. 221 (1), 182-188 (1994).</li><li>Wang, Z., Zhang, F., Dordick, J. S., Linhardt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mass+characterization+of+glycosaminoglycans+with+different+degrees+of+sulfation+in+bioengineered+heparin+process+by+size+exclusion+chromatography.">Molecular mass characterization of glycosaminoglycans with different degrees of sulfation in bioengineered heparin process by size exclusion chromatography.</a> Current Analytical Chemistry. 8 (4), 506-511 (2012).</li><li>Pepi, L. E., Sanderson, P., Stickney, M., Amster, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+mass+spectrometry+for+glycosaminoglycan+analysis:+A+review.">Developments in mass spectrometry for glycosaminoglycan analysis: A review.</a> Molecular and Cellular Proteomics. , 100025 (2021).</li><li>Whiteman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantitative+measurement+of+Alcian+Blue-glycosaminoglycan+complexes.">The quantitative measurement of Alcian Blue-glycosaminoglycan complexes.</a> Biochemical Journal. 131 (2), 343-350 (1973).</li><li>Yuan, 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=Molecular+mass+dependence+of+hyaluronan+detection+by+sandwich+ELISA-like+assay+and+membrane+blotting+using+biotinylated+hyaluronan+binding+protein.">Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein.</a> Glycobiology. 23 (11), 1270-1280 (2013).</li></ol>

GAGs play a central role in many diverse biological processes. One of the principal functions of sulfated GAGs (such as HS and CS) is to interact with and bind to ligands, which can alter downstream signaling functions. An important determinant of GAG binding affinity to cognate ligands is the length of the GAG polymer chain 8,9,14. For this reason, it is important for researchers to be able to define with reasonable precision t...

Glycosaminoglycans (GAGs) are&#160;a diverse family of linear polysaccharides that are a ubiquitous element in living organisms and contribute to many basic physiological processes1. GAGs such as heparan sulfate (HS) and chondroitin sulfate (CS) may be sulfated at distinct positions along the polysaccharide chain, imparting geographic domains of negative charge. These GAGs, when tethered to cell-surface proteins known as proteoglycans, project into the extracellular space and bind to cognate ligands, allowing for the regulation of both cis- (ligand attached to the same cell) and trans- (ligand attached to neighboring cell) signaling processes2. Furthermore, GAGs also perform critical roles as structural elements in tissues such as the glomerular basement membrane3, the vascular endothelial glycocalyx4 and pulmonary epithelial glycocalyx5, and in connective tissues such cartilage6.
The length of GAG polysaccharide chains varies substantially according to its biological context and can be dynamically lengthened, cleaved, and modified by a highly complex enzymatic regulatory system7. Importantly, the length of GAG polymer chains contributes substantially to their binding affinity for ligands and, subsequently, to their biological function8,9. For this reason, determination of the function of an endogenous GAG requires appreciation of its size. Unfortunately, unlike proteins and nucleic acids, very few readily available techniques exist to detect and measure GAGs, which has historically resulted in relatively limited investigation into the biological roles of this diverse polysaccharide family.
This article describes how to isolate and purify GAGs from most biological tissues, and, also, describes how to use polyacrylamide gel electrophoresis (PAGE) to evaluate the length of the isolated polymers with a fair degree of specificity. In contrast to other, highly complex (and often mass spectrometry-based) glycomic approaches, this method can be employed using standard laboratory equipment and techniques. This practical approach may, therefore, expand investigators&#39; ability to determine the biological role of native GAGs and their interaction with contextually important ligands.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accuspin Micro17 benchtop microcentrifuge</td>
			<td>thermoFisher Scientific</td>
			<td>13-100-675</td>
			<td>Any benchtop microcentrifuge/rotor combination capable of 14000 xG is appropriate</td>
		</tr>
		<tr>
			<td>Acrylamide (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP170-100</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Actinase E</td>
			<td>Sigma Aldrich</td>
			<td>P5147</td>
			<td>Protease mix from S. griseus</td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>AC400460100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>A639-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A669S-500</td>
			<td>certified ACS</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP179-25</td>
			<td>electrophoresis grade</td>
		</tr>
		<tr>
			<td>Barnstead GenPure Pro Water Purification System</td>
			<td>ThermoFisher Scientific</td>
			<td>10-451-217PKG</td>
			<td>Any water deionizing/ purification system is an acceptable substitute</td>
		</tr>
		<tr>
			<td>Boric acid (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>A73-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Bromphenol blue (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>B392-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium acetate (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>18-609-432</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Calcium chloride (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>AC349610250</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)</td>
			<td>ThermoFisher Scientific</td>
			<td>28299</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondroitinase ABC</td>
			<td>Sigma Aldrich</td>
			<td>C3667</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion empty cassette for PAGE (1.0mm thick, 12+2 wells)</td>
			<td>Bio-Rad</td>
			<td>3459901</td>
			<td>Any 1.0mm thick PAGE casting cassette system will suffice</td>
		</tr>
		<tr>
			<td>Criterion PAGE Cell system (cell and power supply)</td>
			<td>Bio-Rad</td>
			<td>1656019</td>
			<td>any comparable vertical gel PAGE system will work)</td>
		</tr>
		<tr>
			<td>Dichloromethane (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>AC610931000</td>
			<td>certified ACS</td>
		</tr>
		<tr>
			<td>EDTA disodium salt (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>02-002-786</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Glacial acetic acid (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A35-500</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Glycine (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>G48-500</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Heparanase I/III</td>
			<td>Sigma Aldrich</td>
			<td>H3917</td>
			<td>From Flavobacterium heparinum</td>
		</tr>
		<tr>
			<td>Heparin derived decasaccharide (dp10)</td>
			<td>galen scientific</td>
			<td>HO10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin derived hexasaccharde (dp6)</td>
			<td>Galen scientific</td>
			<td>HO06</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin derived oligosaccharide (dp20)</td>
			<td>galen scientific</td>
			<td>HO20</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A466-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7752020</td>
			<td>Any lyophilizer that can achieve -40C and 0.135 Torr will work; can also be replaced with rotational vacuum concentrator</td>
		</tr>
		<tr>
			<td>Methanol (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A412-500</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Molecular Imager Gel Doc XR System</td>
			<td>Bio-Rad</td>
			<td>170-8170</td>
			<td>Any comparable gel imaging system is an acceptable substitute</td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP171-25</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Phenol red (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>P74-10</td>
			<td>Free acid</td>
		</tr>
		<tr>
			<td>Q Mini H Ion Exchange Column</td>
			<td>Vivapure</td>
			<td>VS-IX01QH24</td>
			<td>Ion exchange column must have minimum loading volume of 0.4mL, working pH of 2-12, and selectivity for ionic groups with pKa of 11</td>
		</tr>
		<tr>
			<td>Silver nitrate (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>S181-25</td>
			<td>certified ACS</td>
		</tr>
		<tr>
			<td>Sodium Acetate (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>S210-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Sodium chloride (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>S271-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>S392-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP220-1</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>TEMED (N,N,N&#39;,N&#39;-tetramethylenediamine)</td>
			<td>thermoFisher Scientific</td>
			<td>BP150-20</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Tris base (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP152-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ultra Centrifugal filters, 0.5mL, 3000 Da molecular weight cutoff</td>
			<td>Amicon</td>
			<td>UFC500324</td>
			<td>Larger volume filter units may be used, depending on sample size.&#160;</td>
		</tr>
		<tr>
			<td>Urea (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacufuge Plus</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td>Any rotational vacuum concentrator will work; can be replaced with lyophilizer</td>
		</tr>
		<tr>
			<td>Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume</td>
			<td>thermoFisher Scientific</td>
			<td>569-0020</td>
			<td>Alternative volumes and filter materials acceptable</td>
		</tr>
	</tbody>
</table>

detection of glycosaminoglycans by polyacrylamide gel electrophoresis and silver staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

Alcian blue is used to stain sulfated GAGs 10; this signal is amplified by use of a subsequent silver stain 11. Figure 1 provides a visual demonstration of the silver staining development process. As demonstrated, the Alcian blue signal representing GAGs separated by electrophoresis is amplified as the developing agent penetrates the polyacrylamide gel. Typically, the developing process will reduce silver and Alcian blue-stained GAGs in a densi...

Watch this Scientific Journal Video about Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining at JoVE.com

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

This protocol describes a simple technique that can be used to detect the presence and measure the length of glycosaminoglycans, which are molecules of increasingly recognized significance to many biological processes. This technique is readily adaptable to most life science laboratories. Comparable glycomic techniques often require equipment and expertise only found in glycochemistry research laboratories.This technique may be especially useful to researchers who are interested in studying the glycocalyx, a polysaccharide coat that lines the endothelial and epithelial surfaces of the human body. Start by placing an empty cassette into the PAGE tank. Cast a resolving gel by mixing 10 milliliters of resolving gel solution with 60 microliters of freshly prepared at 10%ammonium persulfate in a 15 milliliter tube.Then add 10 microliters of TEMED. Invert the tube gently two to three times. Quickly add 10 milliliters of the activated polyacrylamide gel solution to the cassette using a pipette.Overlay with two milliliters of deionized filtered water, and allow the resolving gel to polymerize for 30 minutes. After the resolving gel has fully polymerized, discard the overlaid water. Cast the stacking gel by mixing three milliliters of stacking gel solution with 90 microliters of freshly prepared 10%ammonium persulfate in a 15 milliliter tube.Then add three microliters of TEMED and invert the tube gently two to three times. Use a pipette to quickly add the stacking gel solution over the solidified resolving gel until the cassette is filled to the brim. Fully insert the comb included with the empty polyacrylamide gel cassette and allow the stacking gel to polymerize for 30 minutes.Once the gel is polymerized, remove the tape strip from the bottom of the cassette, and place the cassette back into the PAGE tank assembly. Fill the upper and lower chambers with the respective buffer solutions. Dissolve dried glycosaminoglycan samples in the minimum necessary volume of deionized filtered water and mix with sample loading buffer in a one-to-one ratio.Load the samples and the HS oligosaccharide ladders into the gel. Prerun the gel for five minutes at 100 volts, then run it at 200 volts for 20 to 100 minutes, depending on the acrylamide percentage of the resolving gel solution. Once the run is complete, disassemble the cassette and carefully extract the gel into a large clean container filled with deionized filtered water.Then discard the water and stain the gel in Alcian blue staining solution for five minutes. Discard the Alcian blue stain, and quickly wash two to three times with deionized filtered water until most of the Alcian blue staining solution has been removed. Stain the gel in silver nitrate staining solution in a fresh clean container for 30 minutes.Then wash two to three times for 30 minutes each in deionized filtered water to fully remove the silver staining solution. Discard the water and add developing solution. Carefully observe the gel for the appearance of bands.Depending on the quality of the stain and the mass of the sample loaded, development can take anywhere from a few seconds to several minutes. As soon as the desired bands are visible, immediately discard the developing solution and wash the gel briefly with STOP solution. The isolated and purified glycosaminoglycans were visualized using Alcian blue and silver staining technique after density dependent resolution by a polyacrylamide gel electrophoresis.As seen in this figure, heparan sulfate oligosaccharides of different polymer lengths were readily detectable using this PAGE based approach. The limit of detection of this method, as low as 0.5 micrograms, shows that the technique is highly sensitive in detecting glycosaminoglycans isolated and purified from biological samples. Of the four different heparin sulfate oligosaccharides visualized using this method, unfractionated heparin was least readily detectable.Due to the polydispersity of unfractionated heparin, the density of each individual band from this sample is lower than the bands produced by an equal mass of purified heparin oligosaccharides. The efficiency of glycosaminoglycan purification from liquid biological samples was assessed using bronchoalveolar lavage samples collected from mice, which originally contained relatively low concentrations of glycosaminoglycans. Additionally, the success of this technique was demonstrated using heparin sulfate isolated from whole lung homogenate, which yielded an ample quantity of isolated heparin sulfate, with the smallest fragments equaling approximately 10 disaccharide subunits in mass.It's very important to use freshly prepared reagents, and to filter every reagent used in this protocol. It's critical to use reagents of the highest purity and quality to successfully perform this technique.

Research

JoVE Journal

Biology

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