polyacrylamide gel electrophoresis: an analytical technique to detect heparan sulfates of varying chain lengths isolated from mouse lung tissue

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

Start by placing an empty cassette into the PAGE tank. Cast the resolving gel by mixing 10 milliliters of resolving gel solution with 60 microliters of freshly prepared 10% ammonium persulfate in a 15-milliliter tube. Then, add 10 microliters of TEMED. Invert the tube gently 2 to 3 times.
Quickly add 10 milliliters of the activated polyacrylamide gel solution to the cassette using a pipette. Overlay with 2 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 3 milliliters of stacking gel solution with 90 microliters of freshly prepared 10% ammonium persulfate in a 15-milliliter tube. Then, add 3 microliters of TEMED, and invert the tube gently 2 to 3 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 has 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 1-to-1 ratio. Load the samples and the HS oligosaccharide ladders into the gel.
Re-run the gel for 5 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.

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1. Polyacrylamide gel electrophoresis of isolated and purified glycosaminoglycans
<ol>
	<li>Prepare solutions necessary for polyacrylamide gel electrophoresis (PAGE) in advance (Table 1).&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160; 
	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>

<table><tbody><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>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>EDTA disodium salt (solid)</td><td>ThermoFisher Scientific</td><td>02-002-786</td><td>Molecular biology grade</td></tr><tr><td>Glycine (solid)</td><td>ThermoFisher Scientific</td><td>G48-500</td><td>Electrophoresis grade</td></tr><tr><td>Hydrochloric acid (liquid)</td><td>ThermoFisher Scientific</td><td>A466-250</td><td></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>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>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><tr><td>Acrylamide (solid)</td><td>ThermoFisher Scientific</td><td>BP170-100</td><td>Electrophoresis grade</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></tbody></table>

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.

Table 1: Solutions required for polyacrylamide gel electrophoresis of purified glycosaminoglycans. All solutions must be filtered (0.22 &#181;m) before use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">NOTE: All solutions must be filtered (0.22&#956;m) before use.</td>
		</tr>
		<tr>
			<td>Solution</td>
			<td>Recipe</td>
			<td>Composition</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td rowspan="4">Resolving gel/lower chamber running buffer (2 L or 4 L)</td>
			<td>Boric acid, MW 61.83</td>
			<td>2L: 12.36 g; 4L: 24.76 g</td>
			<td>Desired concentration: 0.1 M</td>
		</tr>
		<tr>
			<td>Tris base, MW 124.14</td>
			<td>2L: 24.2 g; 4L:&#160;</td>
			<td>Desired concentration: 0.1 M</td>
		</tr>
		<tr>
			<td>Disodium EDTA MW 336.21 or dihydrate, MW 372.36</td>
			<td>2L: 6.7 g or 7.4 g, respectively; 4L 13.4 g or 14.8 g, respectively</td>
			<td>Desired concentration: 0.01 M</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>2L or 4L</td>
			<td>Adjust pH to 8.3 after fully dissolving reagents</td>
		</tr>
		<tr>
			<td rowspan="3">Upper chamber running buffer (1 L)</td>
			<td>Glycine</td>
			<td>93 g</td>
			<td>Desired concentration: 1 M</td>
		</tr>
		<tr>
			<td>Tris base, MW 124.14</td>
			<td>24.2 g</td>
			<td>Desired concentration: 0.2 M</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>1 L</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="4">Resolving gel, 22% total acrylamide (500mL)</td>
			<td>Acrylamide, MW 71.08</td>
			<td>100.1 g</td>
			<td>Desired concentration: 20.02% w/v</td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide (bis, MW 154.17)</td>
			<td>10 g</td>
			<td>Desired concentration: 2% w/v</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>75 g</td>
			<td>Desired concentration: 15% w/v</td>
		</tr>
		<tr>
			<td>Resolving gel buffer</td>
			<td>500 mL</td>
			<td>Bring to a total volume of 500mL; will require less than 500mL of buffer total</td>
		</tr>
		<tr>
			<td rowspan="4">Resolving gel, 15% total acrylamide (400mL)</td>
			<td>Acrylamide, MW 71.08</td>
			<td>56.3 g</td>
			<td>Desired concentration: 14.08% w/v</td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide (bis, MW 154.17)</td>
			<td>3.7 g</td>
			<td>Desired concentration: 2% w/v</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>20.8 g</td>
			<td>Desired concentration: 15% w/v</td>
		</tr>
		<tr>
			<td>Resolving gel buffer</td>
			<td>400 mL</td>
			<td>Bring to a total volume of 400mL; will require less than 400mL of buffer total</td>
		</tr>
		<tr>
			<td rowspan="3">Stacking gel (100 mL)</td>
			<td>Acrylamide, MW 71.08</td>
			<td>4.75 g</td>
			<td>Desired concentration: 4.75% w/v</td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide (bis, MW 154.17)</td>
			<td>0.25 g</td>
			<td>Desired concentration: 0.25% w/v</td>
		</tr>
		<tr>
			<td>Resolving gel buffer</td>
			<td>100 mL</td>
			<td>Add 80mL buffer and full dissolve reagents. Then adjust pH to 76.3 with hydrochloric acid, dropwise. Then bring to a total volume of 100 mL with resolving gel buffer.&#160;</td>
		</tr>
		<tr>
			<td rowspan="4">Sample loading buffer (500 mL)&#160;</td>
			<td>Sucrose</td>
			<td>250g</td>
			<td>Desired concentration: 50% w/v</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>500mg</td>
			<td>Desired concentration: 1mg/mL</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>250mg</td>
			<td>Desired concentration: 0.5mg/mL</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>500mL</td>
			<td>Bring to a total volume of 500mL; will require less than 500mL of water total</td>
		</tr>
		<tr>
			<td rowspan="5">Heparin derived oligosacharide &#39;ladder&#39; (2mL each)</td>
			<td>dp6</td>
			<td>0.1mg</td>
			<td rowspan="3">NOTE: Recommend using heparin derived oligosaccharides&#160; 6 polymer subunits in length (aka degree of polymerization 6, or dp6) for the smallest&#160; band, dp20 for the largest&#160; band, and dp10 for middle band. However, other combinations can be used if desired. Desired concentration: 0.05 mg/mL</td>
		</tr>
		<tr>
			<td>dp10</td>
			<td>0.1mg</td>
		</tr>
		<tr>
			<td>dp20</td>
			<td>0.1mg</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>3mL</td>
			<td>Dissolve each oligosaccharide in separate tubes with&#160; 1mL each</td>
		</tr>
		<tr>
			<td>Sample loading buffer</td>
			<td>3mL</td>
			<td>Add 1mL (1:1 mixture) to each oligosaccharide solution</td>
		</tr>
	</tbody>
</table>

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

Sulfated glycosaminoglycans like heparan sulfates, HSs, are negatively charged, linear, sulfated polysaccharides of variable chain lengths, attached to cell surface proteins.
To estimate different-length HSs isolated from mouse lung tissue, begin with gel cassette assembly. Partially fill it with highly concentrated acrylamide-bisacrylamide solution, including catalyzing agents - ammonium persulfate and tetra-methyl-ethylene-diamine. Overlay with water to prevent air contact, which may inhibit polymerization. Incubate.
Catalyzing agents induce acrylamide-bisacrylamide polymerization, forming small-pore resolving gel. Discard water. Add low concentration of acrylamide-bisacrylamide solution with catalyzing agents. Insert a gel comb to create wells. The solution polymerizes, forming large-pore stacking gel.
Load the electrophoresis tank&#39;s chambers with running buffer for optimum electrical conductivity during electrophoresis.
Dissolve the HS mixture in loading buffer containing tracking dye and sucrose. Next, transfer this mixture and standard HS ladders into cassette wells. Sucrose confines the mixture to the wells. Dye helps monitor electrophoresis progression.
Initiate electrophoresis at low voltage. Negatively charged HSs traverse the stacking gel, moving towards the positively charged anode, and concentrate at the stacking-resolving gel interface due to pore size difference.
Increase the voltage. Small-chain HSs migrate quickly through the resolving gel, traversing farther, while long-chain HSs get trapped within the gel matrix and move slowly.
Post electrophoresis, stain the gel. HSs appear as distinct bands, with small-chain HSs positioned lower than long-chain HSs. Length of individual HSs can be determined by comparing with HS standards.

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

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