This protocol describes a simple technique that can be used to detect the presence and measure the length of glycosaminoglycans, which are molecules of increasingly recognized significance to many biological processes. This technique is readily adaptable to most life science laboratories. Comparable glycomic techniques often require equipment and expertise only found in glycochemistry research laboratories.
This technique may be especially useful to researchers who are interested in studying the glycocalyx, a polysaccharide coat that lines the endothelial and epithelial surfaces of the human body. Start by placing an empty cassette into the PAGE tank. Cast a resolving gel by mixing 10 milliliters of resolving gel solution with 60 microliters of freshly prepared at 10%ammonium persulfate in a 15 milliliter tube.
Then add 10 microliters of TEMED. Invert the tube gently two to three times. Quickly add 10 milliliters of the activated polyacrylamide gel solution to the cassette using a pipette.
Overlay with two 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 three milliliters of stacking gel solution with 90 microliters of freshly prepared 10%ammonium persulfate in a 15 milliliter tube.
Then add three microliters of TEMED and invert the tube gently two to three 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 is 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 one-to-one ratio.
Load the samples and the HS oligosaccharide ladders into the gel. Prerun the gel for five 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. Once the run is complete, disassemble the cassette and carefully extract the gel into a large clean container filled with deionized filtered water.
Then discard the water and stain the gel in Alcian blue staining solution for five minutes. Discard the Alcian blue stain, and quickly wash two to three times with deionized filtered water until most of the Alcian blue staining solution has been removed. Stain the gel in silver nitrate staining solution in a fresh clean container for 30 minutes.
Then wash two to three times for 30 minutes each in deionized filtered water to fully remove the silver staining solution. Discard the water and add developing solution. Carefully observe the gel for the appearance of bands.
Depending on the quality of the stain and the mass of the sample loaded, development can take anywhere from a few seconds to several minutes. As soon as the desired bands are visible, immediately discard the developing solution and wash the gel briefly with STOP solution. The isolated and purified glycosaminoglycans were visualized using Alcian blue and silver staining technique after density dependent resolution by a polyacrylamide gel electrophoresis.
As seen in this figure, heparan sulfate oligosaccharides of different polymer lengths were readily detectable using this PAGE based approach. The limit of detection of this method, as low as 0.5 micrograms, shows that the technique is highly sensitive in detecting glycosaminoglycans isolated and purified from biological samples. Of the four different heparin sulfate oligosaccharides visualized using this method, unfractionated heparin was least readily detectable.
Due to the polydispersity of unfractionated heparin, the density of each individual band from this sample is lower than the bands produced by an equal mass of purified heparin oligosaccharides. The efficiency of glycosaminoglycan purification from liquid biological samples was assessed using bronchoalveolar lavage samples collected from mice, which originally contained relatively low concentrations of glycosaminoglycans. Additionally, the success of this technique was demonstrated using heparin sulfate isolated from whole lung homogenate, which yielded an ample quantity of isolated heparin sulfate, with the smallest fragments equaling approximately 10 disaccharide subunits in mass.
It's very important to use freshly prepared reagents, and to filter every reagent used in this protocol. It's critical to use reagents of the highest purity and quality to successfully perform this technique.
