The overall goal of this experiment is to characterize plant cell wall polysaccharides. This method can help us answer key questions in the plant cell wall biology field including glucomannan structure and quantity. The main advantage of this technique is that is doesn't require specialized training or expensive equipment.
Generally, individuals new to this method will struggle with interpretation of the gels, so inclusion and understanding of all the controls described is critical. The alcohol and soluble residue, or AIR, is previously prepared from harvested plant material as described in the text protocol. Weigh six milligrams of AIR into a 15-milliliter centrifuge tube and add water to a final concentration of two milligrams per milliliter.
Vortex to achieve an even suspension. Aliquot 500 micrograms into microfuge tubes. Dry aliquots using a vacuum centrifuge at 30 degrees Celsius overnight.
Pretreat the AIR including an aliquot for a no-enzyme AIR control with one molar sodium hydroxide for one hour at room temperature. After one hour, add 200 microliters of water and 20 microliters of one molar hydrochloric acid to each tube to neutralize the sodium hydroxide. Test that the pH is about six to seven by removing one microliter and spotting it onto the paper pH indicator strips.
Add 50 microliters of one molar ammonium acetate buffer pH 6.0, and add water to a total volume of 500 microliters to each tube. To begin this procedure, add a predetermined amount of mannanases to the AIR aliquots and buffer. Include appropriate negative controls as well as a positive control.
Vortex and then spin briefly to collect the reaction mixture in the bottom of the tube. Incubate at 37 degrees Celsius with gentle agitation overnight. On the following day, stop the reaction by incubating at 95 degrees Celsius for 20 minutes.
Centrifuge at maximum speed for 10 minutes. Save the supernatant and resuspend each pellet in 250 microliters of water. Centrifuge again and retain the supernatant.
Combine both supernatants and dry in a vacuum centrifuge at 30 degrees Celsius for about three hours. Prior to the derivatization procedure, prepare the necessary reagents as described in the text protocol. Warm the stock solution of 0.2 molar ANTS to 60 degrees Celsius to completely dissolve the solid.
To each sample, add five microliters of ANTS, five microliters of two-picoline-borane and 10 microliters of derivatization buffer. Spin briefly to collect in the bottom of the tube, vortex thoroughly and then spin briefly again. Incubate the samples overnight at 37 degrees Celsius protected from light.
On the following day, dry the samples in a vacuum centrifuge at 30 degrees Celsius for about two hours. Resuspend each sample in oligosaccharide standard and 100 microliters of three molar urea. Store at minus 20 degrees Celsius protected from light until required.
Assembly of the gel casting equipment will depend on the brand. For the equipment used in this demonstration, one gel equate to 50 milliliters. In a 50-milliliter centrifuge tube, mix 20.2 milliliters of water, five milliliters of 10x PACE buffer, 24.6 milliliters of 40%acrylamide/Bis-acrylamide, 200 microliters of APS and 20 microliters of TEMED.
Invert gently to mix being careful not to introduce air bubbles. Use a serological or other large-volume pipette to pour the resolving gel to approximately four centimeters below the top of the glass plates. Carefully overlay the gel with isopropyl alcohol.
Allow the gel to polymerize for 20 to 30 minutes. Pour off the top layer. Dry any excess liquid using blotting paper.
Next, make the stacking gel. In a 15-milliliter centrifuge tube, mix 6.8 milliliters of water, one milliliter of 10x PACE buffer, 2.8 milliliters of acrylamide/Bis-acrylamide, 80 microliters of APS and eight microliters of TEMED. Invert to mix, and overlay on top of the polymerized resolving gel.
Gently insert the comb avoiding trapping air bubbles under the comb teeth. After the gel has polymerized, wrap it in moist tissue and then in plastic wrap, and store at four degrees Celsius until required. To assist in keeping track of the loading order and in identifying where the wells are once the comb is removed, use a permanent marker to label the well positions on the glass.
Remove the comb. Fill the wells with one-X PACE buffer. Use a 10-microliter microsyringe to load two microliters of standards and samples into the wells.
Avoid using the outermost lanes which tend to run samples poorly and leave an empty lane in between samples. Assemble the upper chamber of the gel-running apparatus and place the gel in a cooled running tank containing one-X PACE buffer. Fill the upper chamber with one-X PACE buffer.
Turn on the power and run the gel at 200 volts for 30 minutes. After 30 minutes, increase the voltage to 1, 000 volts for one hour and 40 minutes. Protect the gel from light.
Start by wiping the gel imaging system with moist lint-free tissue to ensure it is dust-free. Remove the gel from the PACE tank and view briefly whilst the gel is still in the glass plates to determine if the dye front is still on the gel. Use a wedge tool to open the gel and whilst the gel is still on one glass plate, use a pizza cutter to remove both the stacking gel and the bottom of the gel if the dye front is still on the gel.
Dispense about five milliliters of water onto the surface of the transilluminator and then transfer the gel directly onto the transilluminator. Using the software, set the filter to UV 605 and turn on the longwave UV transilluminator. Take several images at various exposure times making sure to turn the UV light off between images to avoid degrading the fluorescence.
Ensure that at least two of the images have no saturated bands. Save the files as high-resolution TIFF images. A representative gel of a standard PACE assay of cell wall mannan content is shown.
Lanes one, two and three show a ladder of commercially-purchased oligosaccharides at five, 10 and 50 picomole concentration respectively. Lane four shows a mannanase digestion of konjac glucomannan which serves as a positive control for both the enzymatic digestion and the derivatization reaction in the presence of the enzyme and buffer salts. Lane five shows the PACE fingerprint of a wild-type Arabidopsis stem.
Lane six shows the PACE fingerprint of the stem from an Arabidopsis plant lacking the major stem mannan synthase. Compared to the wild-type, only a small quantity of mannan is present as evidenced by the reduced band intensities for all mannanase-derived oligosaccharides. Lane 10 shows the PACE fingerprint of pinewood which contains galactoglucomannan and has a pattern of released oligosaccharides clearly different from that of Arabidopsis.
Lane seven, eight, nine and 11 are various negative controls that reveal nonspecific bands marked by asterisks that should be excluded from the analysis. After watching this video, you should have a really good understanding of how to analyze the structure of mannan using PACE. Once mastered, this technique can be applied to other polysaccharides of interest using different glycosyl hydrolases.
We've had success applying this technique to many other plant species in addition to Arabidopsis as well as to other non-plant species such as yeast mannan.
