Our research focuses on the occurrence, functions, and structures of complex glycans within diverse domestic, industrial, and commercial systems. Further refinements to non-contact microarray technology and our ability to rapidly access sequence information for carbohydrate binding modules is increasing our capacity to both print and interrogate glycan microarrays. Glycans are challenging to study due to their structural heterogeneity, complexity, and variability in biosynthesis.
Many glycan analysis methods necessitate the breakdown of glycans into their constituent monosaccharides. MAPP offers a versatile glycan analysis platform, which retains a lot of the important structural 3D information. To begin, use a mechanical tissue lyser to shake previously prepared AIR-CDTA mixture at 27 hertz for 2 minutes, followed by 10 hertz for 2 hours.
Next, centrifuge the mixture at 10, 000 G for 10 minutes at 4 degrees Celsius. Transfer the supernatant to a sterile microcentrifuge tube. Finally, add sodium hydroxide and sodium borohydride mixture to the residual pellet.
After shaking and centrifuging the pellet, wash the residual pellet 2-3 times with distilled water. Add 30 microliters per milligram of cellulase to the pellet. Then incubate the tubes in a heat block at the enzyme's optimal temperature for 16 hours.
Centrifuge the samples and store the supernatant on a rotary shaker. Centrifuge all stored extracts again at 10, 000 G for 10 minutes at 4 degrees Celsius. Begin by preparing a 1:20 dilution of black Indian ink and glycerol system buffer, then centrifuge for 10 minutes at 15, 000 G at room temperature.
Add 40 microliters of ink solution and glycerol system buffer to the first section of the 384-well plate. Now add 25 microliters of glycerol system buffer to all dilution 1 wells, and 40 microliters to all dilution 2, 3, and 4 wells. Add 25 microliters of extracted glycan sample to the dilution 1 wells to dilute the samples in a 1:1 ratio.
Serially dilute each sample four times by transferring 10 microliters from the dilution 1 to the dilution 2 well then gently mix using a pipette. Repeat the process by transferring 10 microliters of the sample from the dilution 2 to the dilution 3 well. After repeating the process for the dilution 4 well, discard 10 microliters from it so that all wells have a final volume of 40 microliters.
Add 40 microliters of ink solution and glycerol system buffer to the final plate. Then cover the plate with an adhesive cover before centrifuging it for 10 minutes at 3, 000 G at room temperature. To print the samples on the nitrocellulose membrane, start by purging the print head and capillaries multiple times with glycerol system buffer.
Initiate a test print run by loading a plate with the buffer alone. To configure the instrument to print directly from the clean buffer reservoir, click on File followed by New, then choose Print Run. Choose the microplate type and change the slide settings accordingly.
Next, change the Mini-array settings before editing the Spot Property settings. Click on the Overview tab to view the plate. Finally, under the Options tab, select Using buffer and Calculate printing time before allocating file locations to save the file.
After saving the location, press on Start Print Run to proceed with the run. While printing extracted glycan samples, program the system as before. Under the Options tab, click on Using source microplates.
Finally, save the unique grid file generated for the printed microarray for downstream analysis. Defined glycan standards were also included in the printed microarrays as positive controls. The MAPP binding profile obtained for the selected glycan standards corresponds to previously reported epitope specificities.
Begin by cutting out identical printed microarrays from the nitrocellulose membrane and placing them in a suitable vessel for probing. To reduce non-specific binding, add MP-TBST blocking buffer to the microarrays and incubate for 1 hour on a rotating shaker. After incubation, replace the buffer with fresh MP-TBST.
Incubate the arrays with monoclonal antibodies and MP-TBST for 2 hours on a rotating shaker. After incubation, remove the molecular probe solution. Submerge the arrays fully in clean TBST.
Immediately replace the buffer with a fresh volume to remove the residual probe solution. Place the arrays on a rotating shaker for 5 minutes. Replace the TBST with a fresh buffer volume and place the arrays on the shaker for another 5 minutes.
Next, incubate the arrays with alkaline phosphatase conjugated antibodies for 2 hours on a rotating shaker. Once incubation is complete and the microarrays have been washed, cover them in nitro blue tetrazolium and BCIP color development solution to chromogenically detect the antibody binding. Terminate the reaction by immersing the array in clean tap water.
Then place the arrays to dry between blotting paper overnight at ambient temperature. The relative abundance of 16 epitopes diagnostic of non-cellulosic plant cell wall polysaccharides were detected by attaching glycan-directed monoclonal antibodies to printed extracts. Most of the extracted glycans were detected within the alkaline sodium hydroxide fraction.
Strong binding signals were recorded for LM10 and LM11, representing xylan and arabinoxylan within the peels of all the mango varieties tested. LM10 and LM11 displayed preferential binding to the garlic root tissue extract and weak binding to the leaf tissue extract LM19, representing partially methyl esterified or unesterified homogalacturonan bound strongly to some mango variety extracts and only to the coffee pulp fractions.
