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.

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Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...