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

In This Article

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

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).
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.
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).
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.
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.
Fill the upper and lower chambers with upper and lower chamber buffer, respectively.
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.
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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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	...