This article details a method for isolating and analyzing heparan sulfates (HSs) from mouse lung tissue using polyacrylamide gel electrophoresis. The process involves gel preparation, sample loading, and electrophoresis to separate HSs based on their chain lengths.
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.
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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