The overall goal of this procedure is to compare cell wall polymer composition across a broad range of plants or tissue types using glycan microarrays. First, harvest the plant tissues of interest. After homogenization prepare alcohol insoluble residues.
Then extract the cell wall glycans sequentially using CDTA and sodium hydroxide. Then print the cell wall glycan extracts in a microarray using a robot. Probe the microarrays with monoclonal antibodies directed to cell wall glycans, and quantify the spot signals on the microarrays.
Ultimately, results can be used to determine the relative abundance of glycan epitopes across a sample set of interest, We developed this microarray method when we recognize the need to provide a global snapshot of cell wall glycan occurrence across multiple plant tissue types simultaneously and detect compositional differences. So the main advantage of this technique over traditional methods, such as monosaccharide analysis, is that this is a microarray based platform where hundreds of samples can be screened simultaneously. This is a rapid and comprehensive method of profiling cell wall composition For each tissue of interest, start with triplicate samples of 100 milligrams fresh weight of plant tissues using liquid nitrogen.
Homogenize the samples to a fine powder, then transfer the homogenous into 10 milliliter plastic conical tubes to prepare cell wall material. Resuspend the homogenous in 10 milliliters of 80%ethanol at room temperature and vortex vigorously for two minutes. Centrifuge the samples at 3, 500 times G and discard the supernatant.
Repeat the 70%ethanol washes at least three times or until the supernatant is clear. Then perform a final wash with 100%acetone. Palate the alcohol insoluble residues and air dry overnight for a fine and homogenous powder sieve the alcohol insoluble residue samples with a 0.4 millimeter squared mesh.
Next weigh 10 milligrams of the alcohol insoluble residue samples into micro tubes that each contain a three millimeter glass bead for mixing. For the extraction of pectins and polymers associated with pectins, add 500 microliters of 50 millimolar CDTA to each sample briefly vortex the tubes and then mix using the tissue izer for three hours at eight hertz. Centrifuge the samples at 12, 000 times.
G.Carefully remove the supernatants and store at four degrees Celsius. Wash the pellets twice with one milliliter of deionized water. Remove all liquid from the tubes from the remaining cell wall pellet.
Proceed to extract cross-linking glycans with 500 microliters of four molar sodium hydroxide with 0.1%sodium hydride. Using the extraction process described earlier, store the supernatants containing cross-linking glycans at four degrees Celsius. Wash the pellets twice in deionized water.
Then using 500 microliters of Cain. Extract residual polymers such as cellulose centrifuge, the supernatants containing extracted cell wall polymers to remove any particulate matter. Then load 50 microliters of each sample into a polypropylene 384 well microtiter plate using a pre-designed custom layout where samples are arranged according to tissue type and extraction type.
Next, dilute the cell wall polymer samples with deionized water to prepare zero five and 25 fold serial dilution. Set parameters such as pin height collection and dwell time and washing steps for the microarray. Control the humidity of the printing chamber at 60%to prevent sample evaporation.
Now start the printing. The robot uses capillary channel pins to print solutions from the sample plate onto a nitrous cellulose membrane that is attached to a flat plate in the machine. Each spot on the array contains 15 nanoliters of solution and is printed in triplicate.
Print identical microarrays next to each other on the membrane and cut into individual arrays. To reduce non-specific binding. Block each microarray in 5%skim milk powder solution at room temperature for two hours.
Add the monoclonal antibodies specific for cell wall glycan, epitopes, and incubate for two hours. Now to remove nonspecific binding, wash the microarrays three times in PBS for five minutes. Next, probe the microarrays with secondary antibody conjugated to horse radish peroxidase for two hours after three five minute PBS washes.
Develop the microarrays using chromogenic or chemiluminescent substrates. Scan the individual microarrays using a high resolution desktop scanner. Save the images as negative 16 bit TIFF files.
Calculate the integral intensity of each spot using explore image processing software fitted with an automated grid tool. Then export the grid data for each microarray as a text file. Manually import the data file into an Excel spreadsheet for analysis.
Average the integral spot intensity across printing replicates and dilutions to obtain a mean spot intensity value for each sample. Plot the relative mean spot intensities between different samples as a heat map using conditional formatting in Excel or online heat mapper. Tools to remove background signal and false positives for each antibody type, correct the data to 100 and impose a 5%cutoff value.
Comprehensive microarray polymer profiling was used to measure the relative abundance of glycans in six tissue types, anther filaments, pollen ovaries, pets staples, and stigma from nicotiana. A lot of flowers here, the microarray was probed with the monoclonal antibody, JIM five, that is specific for an epitope on peptic polysaccharides. Note, the JIM five epitope is detected in CDTA extracts of all flower tissues.
However, it is highest in pollen and lowest in stigma tissue. Interestingly, the JIM five epitope is also detected in the sodium hydroxide extracts of pollen, but not of other tissues. Quantitative information is obtained by generating standard curves of the different purified polysaccharides that were processed as internal standards on the microarray in ovary tissue.
Approximately 157 micrograms of hoog containing the JIM five epitope was present in each sample accounting for approximately 1.5%of total cell wall residues, whereas 625 micrograms of hoog Ronin containing the JIM five epitope was present in pollen tissue accounting for approximately 6.25%of the total cell wall residue. The relative abundance of 15 glycan epitopes across all tissue types are summarized as a heat map. The intensity of color bars is proportional to the adjusted mean spot intensity for each sample.
A pictorial representation of this data allows rapid interpretation of differences in glycan epitope occurrence between the different flower tissues. These results indicate that individual glycan epitopes are uniquely distributed amongst and a lot of flower types. For example, the LM 13 epitope is most abundant in anthrop filaments and SE L tissues compared to other tissues.
This pattern is in striking contrast to that of shorter chain epitopes preferentially detected by MAB LM six. Cross-linking glycans also have distinct patterns of occurrence in n lot of flowers. For instance, the LM 10 epitope is abundant in stigma tissue compared to other tissues, but is absent from ovary tissue.
The LM 15 xog glycan epitopes are highest in petal and anther filament compared to other tissues, whereas hetero manin are highest in an anthrop filaments. After watching this video, you should have a good understanding on how to extract and determine the relative abundance of cell wall glycans across a broad range of plants and tissue types. Using microarrays once mastered, this technique can be completed in three days.
It's important to remember to treat samples and replicates consistently at each step following this procedure. Other methods like acetic nitric acid assays can be performed to ask additional questions. For example, what is the absolute cellulose content remaining after sequential extraction of pectins and cons glycans using CDTA and sodium hydroxide?
This technique has broad applicability in the field of plant cell biology to rapidly compare glycan occurrence in model plants such as Arabidopsis and Fisca, Nutella, as well as others such as cotton, wheat, eucalyptus, and tobacco.
