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

Abstract

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

This video demonstrates the separation procedure of heparan sulfates isolated from mouse lung tissue using polyacrylamide gel electrophoresis.

Protocol

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 ....

Results

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	.......

Materials

List of materials used in this article

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