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

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1. Polyacrylamide gel electrophoresis of isolated and purified glycosaminoglycans

1. 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 (<20 disaccharide subunits in length).
2. 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 μL of 10% ammonium persulfate (must be freshly prepared), and 10 μL 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.
3. 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 μL of 10% ammonium persulfate (must be freshly prepared), 3 μL of TEMED (add TEMED last).
4. 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.
5. 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.
6. Fill the upper and lower chambers with upper and lower chamber buffer, respectively.
7. 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 "ladders" (see Table 1) into the gel.
8. 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.

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結果

Table 1: Solutions required for polyacrylamide gel electrophoresis of purified glycosaminoglycans. All solutions must be filtered (0.22 µm) before use.

NOTE: All solutions must be filtered (0.22μm) before use.
Solution	Recipe	Composition	Comments
Resolving gel/lower chamber running buffer (2 L or 4 L)	Boric acid, MW 61.83	2L: 12.36 g; 4L: 24.76 g	Desired concentration: 0.1 M
	Tris base, MW 124.14	2L: 24.2 g; 4L:	Desired concentration: 0.1 M
	Disodium EDTA MW 336.21 or dihydrate, MW 372.36	2L: 6.7 g or 7.4 g, respectively; 4L 13.4 g or 14.8 g, respectively	Desired concentration: 0.01 M
	Deionized water	2L or 4L	Adjust pH to 8.3 after fully dissolving reagents
Upper chamber running buffer (1 L)	Glycine	93 g	Desired concentration: 1 M
	Tris base, MW 124.14	24.2 g	Desired concentration: 0.2 M
	Deionized water	1 L
Resolving gel, 22% total acrylamide (500mL)	Acrylamide, MW 71.08	100.1 g	Desired concentration: 20.02% w/v
	N,N'-methylene-bis-acrylamide (bis, MW 154.17)	10 g	Desired concentration: 2% w/v
	Sucrose	75 g	Desired concentration: 15% w/v
	Resolving gel buffer	500 mL	Bring to a total volume of 500mL; will require less than 500mL of buffer total
Resolving gel, 15% total acrylamide (400mL)	Acrylamide, MW 71.08	56.3 g	Desired concentration: 14.08% w/v
	N,N'-methylene-bis-acrylamide (bis, MW 154.17)	3.7 g	Desired concentration: 2% w/v
	Sucrose	20.8 g	Desired concentration: 15% w/v
	Resolving gel buffer	400 mL	Bring to a total volume of 400mL; will require less than 400mL of buffer total
Stacking gel (100 mL)	Acrylamide, MW 71.08	4.75 g	Desired concentration: 4.75% w/v
	N,N'-methylene-bis-acrylamide (bis, MW 154.17)	0.25 g	Desired concentration: 0.25% w/v
	Resolving gel buffer	100 mL	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.
Sample loading buffer (500 mL)	Sucrose	250g	Desired concentration: 50% w/v
	Phenol red	500mg	Desired concentration: 1mg/mL
	Bromophenol blue	250mg	Desired concentration: 0.5mg/mL
	Deionized water	500mL	Bring to a total volume of 500mL; will require less than 500mL of water total
Heparin derived oligosacharide 'ladder' (2mL each)	dp6	0.1mg	NOTE: Recommend using heparin derived oligosaccharides  6 polymer subunits in length (aka degree of polymerization 6, or dp6) for the smallest  band, dp20 for the largest  band, and dp10 for middle band. However, other combinations can be used if desired. Desired concentration: 0.05 mg/mL
	dp10	0.1mg
	dp20	0.1mg
	Deionized water	3mL	Dissolve each oligosaccharide in separate tubes with  1mL each
	Sample loading buffer	3mL	Add 1mL (1:1 mixture) to each oligosaccharide solution

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資料

Name	Company	Catalog Number	Comments
Ammonium persulfate (solid)	ThermoFisher Scientific	BP179-25	Electrophoresis grade
Barnstead GenPure Pro Water Purification System	ThermoFisher Scientific	10-451-217PKG	Any water deionizing/ purification system is an acceptable substitute
Boric acid (solid)	ThermoFisher Scientific	A73-500	Molecular biology grade
Bromphenol blue (solid)	ThermoFisher Scientific	B392-5
Criterion empty cassette for PAGE (1.0mm thick, 12+2 wells)	Bio-Rad	3459901	Any 1.0mm thick PAGE casting cassette system will suffice
Criterion PAGE Cell system (cell and power supply)	Bio-Rad	1656019	Any comparable vertical gel PAGE system will work)
EDTA disodium salt (solid)	ThermoFisher Scientific	02-002-786	Molecular biology grade
Glycine (solid)	ThermoFisher Scientific	G48-500	Electrophoresis grade
Hydrochloric acid (liquid)	ThermoFisher Scientific	A466-250
N,N'-methylene-bis-acrylamide (solid)	ThermoFisher Scientific	BP171-25	Electrophoresis grade
Phenol red (solid)	ThermoFisher Scientific	P74-10	Free acid
Sucrose (solid)	ThermoFisher Scientific	BP220-1	Molecular biology grade
TEMED (N,N,N',N'-tetramethylenediamine)	ThermoFisher Scientific	BP150-20	Electrophoresis grade
Tris base (solid)	ThermoFisher Scientific	BP152-500	Molecular biology grade
Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume	ThermoFisher Scientific	569-0020	Alternative volumes and filter materials acceptable
Acrylamide (solid)	ThermoFisher Scientific	BP170-100	Electrophoresis grade
Heparin derived decasaccharide (dp10)	Galen scientific	HO10
Heparin derived hexasaccharde (dp6)	Galen scientific	HO06
Heparin derived oligosaccharide (dp20)	Galen scientific	HO20