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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Biological Techniques

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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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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'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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