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