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>
		<li>Place each tissue sample into a small glass container with a lid for delipidation. 
		NOTE: Sample mass may vary per tissue of interest and experimental needs. 50 mg or less is typically sufficient for adequate GAG yield, but some optimization may be required by the end-user.&#160;</li>
		<li>Add 500 &#956;L of the methanol:dichloromethane solution to each glass container and mix. Ensure all solid tissue samples are completely submerged in solvent. 
		NOTE: Use serological pipettes or plastic conical tubes to mix and handle the methanol/dichloromethane solution; other plastics may dissolve.</li>
		<li>Place samples on a shaker (in secured rack) in a chemical fume hood and agitate gently for 1 h.</li>
		<li>&#160;Agitate the samples to mix and centrifuge at 17,000 x g for 5 min at 4 &#176;C.</li>
		<li>Carefully pipette out the organic solvent fraction (supernatant). This is the lipid fraction - it does not contain GAGs and can be discarded. 
		NOTE: Try not to disturb the tissue as small pieces could be lost. It is preferable to leave some of the organic layer in the sample container, rather than risking the sample loss.</li>
		<li>Leave the lid of the glass container open and let it evaporate overnight in the hood. Change the tube for the next step as those&#160;tubes will not survive further processing.</li>
		<li>Once the solvent mixture has fully evaporated, proceed to mechanical disintegration (if residual sample is &#62;50 mg) or Actinase E digestion (&#60; 50 mg).&#160;</li>
	</ol>
	</li>
	<li>Mechanical disintegration of solid tissue (optional; for larger tissue samples) 
	NOTE: Most smaller pieces of solid tissue (approximately 50 mg or less) should dissolve completely during the digestion step. However, larger samples will require mechanical disintegration.
	<ol>
		<li>Flash-freeze samples of interest by placing them in an appropriately sized polypropylene tube and placing the closed tube into liquid nitrogen. Allow the sample to freeze until completely solid.</li>
		<li>Using a clean mortar and pestle, grind frozen samples into a powder-like consistency.</li>
		<li>Proceed directly to Actinase E digestion (Step 1.4).</li>
	</ol>
	</li>
	<li>Sample desalting and concentration 
	NOTE: This is an optional step and only required for dilute liquid tissue samples (e.g., broncho-alveolar lavage fluid (BALF) or plasma).
	<ol>
		<li>Pool liquid samples into appropriate experimental groups (e.g., by biological replicate, or experimental group)</li>
		<li>Place total sample volume into a 500 &#956;L centrifugal filter column with a molecular weight cut-off (MWCO) of 3,000 Da.</li>
		<li>Spin for 30 min at 14,000 x g at room temperature. Repeat as needed if the desired sample volume exceeds capacity of the centrifugal filter column.</li>
		<li>Wash each column 3x with 400 &#956;L of deionized, filtered water. Discard the flow through.</li>
		<li>Invert the filter and spin for 1 min at 2,000 x g in fresh, appropriately labeled collection tubes. Freeze at -80 &#176;C or proceed to next step.</li>
	</ol>
	</li>
	<li>Actinase E digestion 
	NOTE: This step is required to digest and ultimately remove protein contaminants from your sample.
	<ol>
		<li>Mix samples 1:1 with recombinant Actinase E to a desired concentration of 10 mg/mL. Add appropriate volume of 10x digestion buffer concentrate. Desired final concentration: 0.005 M calcium acetate and 0.01 M sodium acetate, pH 7.5. For example: 190 &#956;L of liquid sample, 190 &#956;L of 20 mg/mL Actinase E, 20 &#956;L of 0.05 M calcium acetate, 0.1 M sodium acetate.</li>
		<li>Agitate samples gently to mix, then digest for 48-72 h at 55 &#176;C (up to 7 days for whole tissue).</li>
		<li>Heat to 80 &#176;C for 15-20 min to heat inactivate Actinase E.&#160;</li>
		<li>Freeze the sample at -80 &#176;C or continue to step 1.5.</li>
	</ol>
	</li>
	<li>Sample desalting and concentration 
	NOTE:&#160;If a low mass of the GAG of interest is anticipated in the biological sample, or if conservation of consumable reagents (i.e., centrifugal filter columns) is desirable, samples can be pooled to produce a single concentrate per experimental&#160;group (e.g., by biological replicate, or experimental group).
	<ol>
		<li>Place total sample volume into a 500 &#956;L centrifugal filter column with a MWCO of 3,000 Da.</li>
		<li>Spin for 30 min at 14,000 x g at room temperature. Repeat as needed, if the desired sample volume exceeds capacity of the centrifugal filter column.</li>
		<li>Wash each column 3x with 400 &#956;L deionized, filtered water. Discard the flow through.</li>
		<li>Invert filter and spin for 1 min at 2,000 x g in fresh, appropriately labeled collection tubes (generally included with the centrifugal filter columns). Freeze at -80 &#176;C or proceed to next step.</li>
	</ol>
	</li>
	<li>Desiccation
	<ol>
		<li>Either place the dissolved samples from step 1.5.5 in a rotational vacuum concentrator overnight or lyophilize the samples as detailed below.</li>
		<li>Freeze samples thoroughly either overnight in -80 &#176;C or by dipping in liquid nitrogen.</li>
		<li>Pierce sample lids with an 18 G needle and place in the lyophilizer chamber. Add paper towels for packing as needed.</li>
		<li>Fix lyophilizer chamber to lyophilizer and freeze dry overnight (at least -40 &#176;C, 0.135 Torr)</li>
	</ol>
	</li>
	<li>Cation exchange column
	<ol>
		<li>Resuspend desiccated samples in up to 400 &#956;L of 8 M urea, 2% CHAPS solution (or, if pooling samples, resuspend to a max of (400/n) &#956;L, where n = the number of samples in desired pool. Use as little of the detergent solution as possible.</li>
		<li>Equilibrate the cation exchange (IEX) column with 400 &#956;L of 8 M urea, 2% CHAPS solution. Spin for 5 min at 2,000 x g at room temperature.</li>
		<li>Load 400 &#956;L of sample/pooled samples into the IEX column. Spin for 5 min at 2,000 x g.</li>
		<li>Wash 3x with 400 &#956;L of 8 M urea, 2% CHAPS solution. Spin for 5 min at 2,000 x g everytime.</li>
		<li>Elute 3x with 400 &#956;L of 0.2 M NaCl. Spin for 5 min at 2,000 x g each. This is the low-affinity fraction - this can be retained for quality control purposes if desired.</li>
		<li>Elute 3x with 400 &#956;L of 2.7 M (16%) NaCl. Spin for 5 min at 2,000 x g each. This fraction will contain the isolated glycosaminoglycans of interest - keep all of it!</li>
		<li>To desalt each eluted fraction, add methanol up to 80 vol% and incubate at 4 &#176;C overnight. Spin each sample for 5 min at 2,000 x g. Recover the solid residue as this is dried de-salted glycosaminoglycan. 
		NOTE: Alternatively, skip this step and proceed to step 1.8 to de-salt the eluate without methanol.</li>
	</ol>
	</li>
	<li>Sample desalting and concentration
	<ol>
		<li>If necessary, pool eluted fractions (from 1.7.6) into appropriate experimental groups (e.g., by biological replicate, or experimental group).</li>
		<li>Place total sample volume into a 500 &#956;L centrifugal filter column with a MWCO of 3,000 Da.</li>
		<li>Spin for 30 min at 14,000 x g at room temperature. Repeat as needed, if the desired sample volume exceeds capacity of the centrifugal filter column.</li>
		<li>Wash each column 3x with 400 &#956;L deionized, filtered water. Discard flow through. Proceed directly to step 1.9.</li>
	</ol>
	</li>
	<li>Chondroitin digestion 
	NOTE: The purpose of this step is to remove GAGs not of interest to the end user. In this case, chondroitinase is used to remove chondroitin. In the tissues used for generating Representative Results (broncho-alveolar lavage fluid and whole lung), digesting chondroitin sulfate leaves heparan sulfate as the primary residual GAG. End users may need to add additional digestion steps depending on their experimental aims.&#160;
	<ol>
		<li>Load 350 &#181;L of digestion buffer (50 mM ammonium acetate with 2 mM calcium chloride adjusted to pH 7.0) to the centrifugal filter column without touching the membrane.</li>
		<li>Add 5 &#181;L of recombinant chondroitinase ABC.</li>
		<li>Place the samples tube into a 37 &#176;C oven and incubate for 1 h.</li>
		<li>Turn the column over and place into appropriately labeled collection tubes. Spin for 1 min at 2000 x g.</li>
		<li>Heat samples to 80 &#176;C for 15-20 min to inactivate chondroitinase ABC.</li>
	</ol>
	</li>
	<li>Sample desalting and concentration
	<ol>
		<li>If necessary, pool chondroitin digested samples from step 1.9.5 into appropriate experimental groups (e.g., by biological replicate, or experimental group)</li>
		<li>Place the total sample volume into a 500 &#956;L centrifugal filter column with a MWCO of 3,000 Da.</li>
		<li>Spin for 30 min at 14,000 x g at room temperature. Repeat as needed if desired sample volume exceeds capacity of the centrifugal filter column.</li>
		<li>Wash each column 3x with 400 &#956;L deionized, filtered water. Discard the flow through.</li>
		<li>Invert filter and spin for 1 min at 2,000 x g in fresh, appropriately labeled collection tubes. Freeze at -80 &#176;C or proceed to next step.</li>
	</ol>
	</li>
	<li>Desiccation 
	NOTE: In this step, desiccation is necessary so that samples can be resuspended in the smallest possible&#160;quantity of water and running buffer.
	<ol>
		<li>Either place the chondroitin digested samples from step 1.10.5 directly in a rotational vacuum concentrator overnight or lyophilize as follows:</li>
		<li>Freeze samples thoroughly either overnight in -80 &#176;C or by dipping in liquid nitrogen.</li>
		<li>Pierce sample lids with an 18 G needle and place in the lyophilizer chamber. Add paper towels for packing as needed.</li>
		<li>Fix lyophilizer chamber to lyophilizer and freeze dry overnight (at least 40 &#176;C, 0.135 Torr)</li>
	</ol>
	</li>
</ol>
2. Polyacrylamide gel electrophoresis of isolated and purified glycosaminoglycans
<ol>
	<li>Prepare solutions necessary for polyacrylamide gel electrophoresis (PAGE) in advance (Table 1). 
	NOTE: Select the percent acrylamide of resolving gel solution depending on the size of the glycosaminoglycans expected to be in the sample. 15% is recommended for resolving larger fragments (greater than 30 disaccharide subunits in length); 22% for smaller fragments (&#60;20 disaccharide subunits in length).</li>
	<li>Place empty cassette into the PAGE tank. Cast the resolving gel as follows: In 15 mL tube, mix 10 mL of resolving gel solution, 60 &#956;L of 10% ammonium persulfate (must be freshly prepared), and 10 &#956;L of TEMED (add TEMED last). Invert the tube gently 2-3x. Use pipette to quickly add the above 10 mL solution to cassette. Overlay with 2 mL of deionized, filtered water and allow the resolving gel to polymerize for 30 min. 
	NOTE: The following PAGE protocol has been optimized for a vertical PAGE system using 13.3 x 8.7cm (width x length) 1.0mm thick casting cassettes with a total volume of approximately 12mL. Other cassette systems can be used but may require optimization by the end-user.</li>
	<li>After the resolving gel has fully polymerized, discard the overlaid water and cast the stacking gel as follows: in a 15 mL tube, mix 3 mL of the stacking gel solution, 90 &#956;L of 10% ammonium persulfate (must be freshly prepared), 3 &#956;L of TEMED (add TEMED last).</li>
	<li>Invert the tube gently 2-3x. Use a pipette to quickly add the stacking gel solution over the solidified resolving gel; fill cassette to brim. Fully insert comb included with the set up. Allow the stacking gel to polymerize for 30 min.</li>
	<li>Once the gel has polymerized, ensure the tape strip is removed from the bottom of the cassette, and place the cassette back into the PAGE tank assembly.</li>
	<li>Fill the upper and lower chambers with upper and lower chamber buffer, respectively.</li>
	<li>Dissolve the dried samples from step 1.11.4 in the minimum necessary volume of deionized, filtered water (at most, 50% of the volume of the wells in the PAGE gel). Mix 1:1 with sample loading buffer. Load the samples and the HS oligosaccharide &#34;ladders&#34; (see Table 1) into the gel.</li>
	<li>Pre-run the gel for 5 min at 100 V. Then run the gel at 200 V for 20-25 min (for a 15% polyacrylamide resolving gel), 40-50 min (for an 18% polyacrylamide resolving gel), 90-100 min (for 22% polyacrylamide resolving gel). 
	NOTE: Some optimization of the 200 V run time may be necessary. Phenol red migrates ahead of heparin oligosaccharides that are 2 polymer subunits in length (i.e., degree of polymerization 2, or dp2); bromophenol blue migrates ahead of dp10-dp14. Best results are obtained when the voltage is applied such that the phenol red band migrates almost, but not quite, to the bottom of the gel. Adjust run time accordingly.</li>
</ol>
3. Silver staining protocol
<ol>
	<li>Prepare all solutions necessary for silver staining in advance (Table 2). 
	NOTE: Do not directly touch the PAGE gel until it has been stained, developed, and placed in stop solution. Instead, manipulate the gel using clean plastic or glass tools. Directly handling the gel will result in finger-print distortions and other visible artifacts on the gel after staining. &#160;</li>
	<li>Once the run is completed, disassemble cassette and extract gel in a clean, medium-large container filled with deionized, filtered water. 
	NOTE: To avoid directly handling the gel, use pipette tip or other plastic object to gently peel the gel away from the cassette while submerged in water. Gel may be fragile - handle carefully.
	<ol>
		<li>Discard the water. Stain the gel in Alcian blue staining solution for 5 min.</li>
		<li>Discard Alcian blue stain. Quickly rinse/wash 2-3x with deionized, filtered water until most of the Alcian blue staining solution has been removed.</li>
		<li>Allow to de-stain in deionized, filtered water overnight on rocker. Ensure that there is ample volume of deionized, filtered water to ensure any residual stain is fully washed off the gel overnight.</li>
		<li>Wash gel in 50% methanol (40 min total, change solution 2-3x).</li>
		<li>Wash gel in deionized, filtered water for 30 min. Discard water and repeat 3 more time for a total of 2 h, replacing the water each time.</li>
		<li>In a fresh, clean container, stain the gel for 30 min in silver nitrate staining solution.</li>
		<li>Quickly rinse/wash 2-3x in deionized, filtered water to fully remove the silver staining solution.</li>
		<li>Wash for 30 min in deionized, filtered water. Discard water and repeat 2x for a total of 90 min, replacing the water bath each time.</li>
		<li>Discard water and add developing solution.</li>
		<li>Once developing solution is added, carefully observe the gel and watch 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.</li>
		<li>As soon as the desired bands are visible, immediately discard developing solution and wash briefly with stop solution.</li>
		<li>Discard the stop solution wash and replace with fresh stop solution. Allow to soak for 1 h on a rocker or shaker.</li>
		<li>Wash in deionized, filtered water overnight (however, the gel can be imaged immediately after stop solution wash).</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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><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&#039;-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&#039;,N&#039;-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.&amp;nbsp;</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...

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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 ...

detection-glycosaminoglycans-polyacrylamide-gel-electrophoresis

Research

JoVE Journal

Biology

4.6K Views.  University of Colorado Anschutz Medical Campus. Dit protocol beschrijft een eenvoudige techniek die kan worden gebruikt om de aanwezigheid te detecteren en de lengte van glycosaminoglycanen te meten, moleculen van steeds meer erkende betekenis voor veel biologische processen. Deze techniek is gemakkelijk aan te passen aan de meeste life science laboratoria. Vergelijkbare glycomische technieken vereisen vaak apparatuur en expertise die alleen te vinden zijn in glycochemische onderzoekslaboratoria.Deze techniek kan vooral nuttig zijn ...

Video: Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Chemistry

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Biochemistry

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Genetics

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Polyacrylamide Gel Electrophoresis: An Analytical Technique to Detect Heparan Sulfates of Varying Chain Lengths Isolated from Mouse Lung Tissue

Source: LaRiviere, W. B. et al., <a href="https://www.jove.com/v/62319">Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining</a>. J. Vis. Exp. (2021)
This video demonstrates the separation procedure of heparan sulfates isolated from mouse lung tissue using polyacrylamide gel electrophoresis.

Source: LaRiviere, W. B. et al., Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (2021)
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

Source: Sultana, S. et al. <a href="https://www.jove.com/v/62628">Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor</a>. J. Vis. Exp. (2021).
This video demonstrates a denaturing polyacrylamide-based separation technique for proteins. This technique helps in the preliminary identification of proteins based on their molecular weights.

Source: Sultana, S. et al. Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor. J. Vis. ...

Fluorescent Silver Staining: A Technique to Visualize Total Protein in Polyacrylamide Gels

Source: Wong, A. Y. H. et al.<a href="https://www.jove.com/v/58669"> Fluorescent Silver Staining of Proteins in Polyacrylamide Gels.</a> J. Vis. Exp. (2019)
In this video, we demonstrate a silver staining method of polyacrylamide gels for the visualization of total protein by using fluorogenic probes.

Source: Wong, A. Y. H. et al. Fluorescent Silver Staining of Proteins in Polyacrylamide Gels. J. Vis. Exp. (2019)
In this video, we demonstrate a silver ...

Staining Techniques

Biomolecule staining

Silver Staining of Sulfated Glycosaminoglycans: A Staining Method for Visualization of Sulfated Glycosaminoglycans in Polyacrylamide Gels

Source: LaRiviere, W. B., et al. <a href="https://www.jove.com/v/62319">Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining.</a> J. Vis. Exp. (2021)
In this video, we demonstrate a procedure for combined Alcian blue and silver staining of sulfated glycosaminoglycan oligosaccharide fragments. This combined staining technique significantly amplifies the bands of glycosaminoglycan polymers of different lengths in polyacrylamide gels.

Source: LaRiviere, W. B., et al. Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (2021)
In this ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

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