The authors would like to acknowledge ArrayJet for their expert advice regarding microarray robotics. SS and JS would like to acknowledge the support from Fundamental Fund 2022 (FF65/004), Chiang Mai University.

The main experimental stages of the MAPP method are summarized in Figure 1.
1. Preparation of samples
NOTE: Here, the method is applied to plant tissues for illustrative purposes. The plants selected were Coffea arabica, Allium sativum var. ophioscorodon, and several Thai mango varieties (Aokrong, Kam, Rad, Chokanan, Mamkamdang, Talabnak, Mahachanok, and Nga). The plants were selected for their commercial importance. Their processing for human consumption generates currently underutilized agro-industrial waste, which may provide a source of value-added products, including pure glycans. Thus, MAPP was applied to characterize the glycan composition of the waste plant biomass for bioprospecting purposes.
<ol>
	<li>Separate the plant material into different tissues (e.g., root, stem, and leaves).</li>
	<li>Dry the plant tissues in a hot air oven at 40 &#176;C for 12-24 h (decrease/increase the time in accordance with the sample). Alternatively, snap freeze the samples in liquid nitrogen and subsequently lyophilize for ~4 days.</li>
	<li>Homogenise the samples to a fine powder using a pestle and mortar or a mechanical tissue lyser (see Table of Materials) with a ball bearing in each tube. 
	&#8203;NOTE: For fresh plant tissue, drying or lyophilizing prior to homogenization is recommended. Generally, homogenization with a pestle and mortar is sufficient for most dry samples. For particularly resilient samples, such as grains, legumes, and processed food items like pasta, snap freezing in liquid nitrogen can increase the speed and efficiency of sample homogenization. We have found that mechanical homogenization is compatible with nearly all sample types, is considerably faster and less labor-intensive, and effectively minimizes the risk of sample cross-contamination from repeated use of the same equipment (i.e., pestle and mortar).</li>
</ol>
2. Preparation of alcohol insoluble residue (AIR)
<ol>
	<li>Add 1.5 mL of 70% (v/v) ethanol to 50-100 mg of air-dried and homogenized sample material.</li>
	<li>Vortex thoroughly to mix and then centrifuge at 10,000 x g for 10 min at room temperature. Discard the resulting supernatant using a pipette and retain the pellet.</li>
	<li>To the remaining pellet, add 1.5 mL of methanol and chloroform (1:1 [v/v]). Vortex, centrifuge, and discard the supernatant, as per step 2.2.</li>
	<li>To the remaining pellet, add 1.5 mL of 100% acetone. Vortex, centrifuge, and discard the supernatant, as per step 2.2.</li>
	<li>Place the resultant pellet either overnight in a fume hood to allow the residual acetone to evaporate or in a vacuum centrifuge until dry. 
	NOTE: AIR material can be stored at ambient temperature until required.</li>
</ol>
3. Glycan extraction
NOTE: If possible, perform all extraction steps in a tissue lyser with a ball bearing in each tube to aid resuspension. If a tissue lyser is unavailable, extractions can be performed instead with continuous stirring or shaking. It may be necessary to extend the extraction time if this is not possible.
<ol>
	<li>To 10 mg of AIR material, add 30 &#181;L/mg of 50 mM cyclohexanediaminetetraacetic acid (CDTA; see Table of Materials), pH 7.5.</li>
	<li>Shake at 27 Hz&#160;for 2 min, followed by 10 Hz&#160;for 2 h.</li>
	<li>Centrifuge at 10,000 x g for 10 min at 4 &#176;C. Retain the resultant supernatant, add it to a sterile microcentrifuge tube, and store it at 4 &#176;C on a rotary shaker.</li>
	<li>To the residual pellet, add 30 &#181;L/mg 4 M NaOH + 0.1 % (w/v) NaBH4. 
	CAUTION: NaBH4 is toxic if swallowed. Use personal protective equipment (PPE). Handle under a fume hood. Avoid dust formation. Avoid breathing dust. Do not allow the product to come into contact with water.</li>
	<li>Repeat steps 3.2 to 3.3. Wash the residual pellet two or three times with dH2O to remove the residual NaOH.</li>
	<li>Add 30 &#181;L/mg cellulase (preferably GH5 endo-1,4-&#946;-glucanase, in appropriate enzyme buffer-as recommended by the manufacturer; see Table of Materials) to the pellet and incubate at the enzyme optimal temperature for 16 h.</li>
	<li>Centrifuge the samples at 10,000 x g for 10 min at 4&#160;&#176;C . Retain the supernatant, transfer to clean microcentrifuge tubes, and store at 4 &#176;C on a rotary shaker. Once extracted, the samples must be printed as soon as possible.</li>
	<li>Centrifuge all the stored extracts again at 10,000 x g for 10 min at 4&#160;&#176;C. 
	&#8203;NOTE: The samples must be free of particulates and debris. Pass through a 0.2 &#181;m spin filter prior to microarray printing if necessary. Particularly viscous samples are incompatible with microarray analysis as the microarrayer capillaries and the print head can become easily clogged. As a general rule, users should be able to pipette all the samples intended for printing with a standard low-volume pipette.</li>
</ol>
4. Preparation of standards
<ol>
	<li>Prepare 1 mg/mL solutions of defined glycan standards (Table 1) in sterile dH2O. If using pachyman as a standard, dissolve in 4 M NaOH instead and neutralize with glacial acetic acid following solubilization.</li>
	<li>Store the prepared standards overnight at 4 &#176;C on a rotary shaker to allow complete solubilization.</li>
	<li>Centrifuge all the standards for 10 min at 10,000 x g at 4&#160;&#176;C to pellet any debris. The resultant supernatant is used for subsequent printing.</li>
</ol>
5. Microarray printing
<ol>
	<li>Prepare a 1:20 (v/v) dilution of black Indian ink/drawing ink in glycerol system buffer (GSB; combine 47% glycerol, 52.9% dH2O, 0.06% Triton X-100, and 0.04% biocide [0.15%-0.17% cupric nitrate and 1.4%-2.0% magnesium nitrate in water] and filter sterilize) (see Table of Materials) and centrifuge for 10 min at 15,000 x g (room temperature). 
	NOTE: Ink solution is necessary to create a top and bottom border around the printed samples, so that the printed microarrays can be visually detected on the membrane. However, the ink solution is likely to contain sediment. All solutions must be free of particulates for printing, so avoid disturbing the sediment when pipetting. Discard and prepare fresh when this is no longer possible. The solution cannot easily be filtered to remove particulates.</li>
	<li>Add 40 &#181;L of ink solution and GSB to the first section of the first 384-well plate (Figure 2).</li>
	<li>Add 25 &#181;L of GSB to all dilution 1 (D1) wells. Add 40 &#181;L of GSB to all dilution 2, 3, and 4 (D2-D4) wells.</li>
	<li>Dilute the extracted glycan samples and defined glycan substrates 1:1 (v/v) with GSB by adding 25 &#181;L of extracted glycan sample to the D1 wells in order.</li>
	<li>Serially dilute each sample four times by taking 10 &#181;L of the sample from the D1 well and adding to the D2 well. Aspirate gently with a pipette to mix.</li>
	<li>Repeat the process by taking 10 &#181;L of sample from the D2 well and adding it to the D3 well.</li>
	<li>Repeat for the D4 well. After mixing, discard 10 &#181;L from the D4 well so that each well contains a final volume of 40 &#181;L.</li>
	<li>Add 40 &#181;L of ink solution and GSB to the final block of the final plate.</li>
	<li>Cover the plates with an adhesive plate cover and centrifuge for 10 min at 3,000 x g (room temperature). Ensure no bubbles remain following centrifugation and repeat if necessary.</li>
	<li>Using a non-contact piezoelectric microarray printing robot, print the samples onto the nitrocellulose membrane (see Table of Materials) following the steps below. 
	NOTE: Below are actions recommended for optimum print quality; however, the specific parameters required will ultimately depend on the instrument used. We advise that users contact the instrument manufacturer to discuss the appropriate customizations and print settings required for their device.
	<ol>
		<li>Prior to printing, empty the waste buffer reservoir and fill the clean buffer reservoir with clean GSB, as necessary. Switch on the instrument and allow it to stabilize for &#62;10 min if it has an integrated humidity and temperature control system. Switch on the microarrayer and initialize the system. 
		NOTE: Running a test to determine the internal pressure of the instrument is recommended. If the pressure is too low, then it may be necessary to perform a high-pressure purge. Again, contact the instrument manufacturer to discuss the specific operation of the specific instrument.</li>
		<li>Purge the print head and capillaries several times with GSB to remove debris and potential contaminants. Perform a test print run by either loading a plate of GSB alone or setting the instrument up to print from the clean buffer reservoir directly, bypassing sample aspiration from a loaded plate. 
		NOTE: It is not necessary to perform the test print using a nitrocellulose membrane; clean microscope slides are sufficient and offer the advantage that the spot size, shape, and quality can be visually assessed prior to sample printing.</li>
		<li>When printing extracted glycan samples, program the system to flush with clean GSB between each sample. 
		NOTE: Typically, the sample volume per printed spot is 100 pL to 10 nL, and the spot size ranges from 20 &#181;m to 100 &#181;m, depending on the sample volume selected. Printing 100 microarrays from one source 384-well plate takes approximately 40 min, including system flushing. The resultant microarrays will be approximately 1 cm2 in size. Additional plates will increase the length of the array by approximately 1 cm per plate. A schematic of the printed microarray design is shown in Figure 3. Once printed, the microarrays are ready to use immediately and can be stored for several years. Should the print job need to be repeated for any reason, due to the potential evaporation of samples during printing, it is recommended that a new sample plate be loaded and the original plate discarded.</li>
		<li>Once a week, thoroughly clean the print head and capillaries. Do this by loading a 384-well containing a 1:20 dilution of concentrated NaOH in GSB and perform&#160;a 40 min to 1 h print run onto microscope slides. Users should ensure this cleaning solution is compatible with their instrument before proceeding.</li>
	</ol>
	</li>
	<li>Save the unique grid file (.gal file) produced for the printed microarray ready for downstream analysis.</li>
</ol>
6. Microarray probing
<ol>
	<li>Cut out individual, identical printed microarrays from the nitrocellulose membrane and place them into an appropriately sized vessel for probing (e.g., a 12- or 24-well microtiter plate) (see Table of Materials). The array should lie&#160;flat on the base of the vessel. One microarray is required per probe and represents one technical replicate.</li>
	<li>To reduce non-specific binding, incubate the microarrays for 1 h in MP-TBST blocking buffer (1x Tris-buffered saline, pH 7.5, + 0.1% [v/v] TWEEN 20 [TBST] and supplemented with 5% [w/v] skimmed-milk powder; see Table of Materials) on a rotating/rocking shaker. Ensure that the volume is sufficient to submerge the entire array.</li>
	<li>Following incubation, remove the MP-TBST and replace it with a fresh volume of MP-TBST.</li>
	<li>Incubate the arrays with monoclonal antibodies (mAbs) or His-tagged CBMs, or other GRMPs (e.g., lectins), diluted 1:10-1:1,000 (as specified by the manufacturer; see Table of Materials) in MP-TBST for 2 h on a rotating/rocking shaker.</li>
	<li>Following incubation, remove the molecular probe solution and cover the arrays in clean TBST, ensuring the arrays are fully submerged. To remove residual probe solution, immediately remove the TBST and replace with a fresh volume. Place the arrays on a rotating/rocking shaker for 5 min. After 5 min, remove the TBST, replace with a fresh volume, and place on a rotating/rocking shaker for 5 min. 
	NOTE: This process should be repeated three times, not including the initial addition and immediate removal of TBST.</li>
	<li>Incubate the arrays with alkaline-phosphatase conjugated secondary antibodies (anti-mouse, anti-rat, anti-rabbit, anti-His, as appropriate; see Table of Materials) diluted 1:1,000 in MP-TBST for 2 h on a rocking/rotating shaker. 
	NOTE: Horseradish peroxidase-conjugated secondary antibodies are also suitable, used in conjunction with tetramethyl benzidine (TMB)/hydrogen peroxide substrate for color development.</li>
	<li>Following incubation, repeat the wash procedure, as per step 6.5, to remove non-specifically bound secondary antibodies.</li>
	<li>Cover the arrays in nitro-blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) color development solution (see Table of Materials) for the chromogenic detection of antibody binding. Leave until purple precipitate spots develop at the antigen binding sites (typically 5-30 min, however the arrays should be monitored closely to avoid oversaturation, as the reaction can occur rapidly). 
	CAUTION: BCIP is harmful when in contact with the skin and may cause respiratory irritation. Use PPE. Avoid dust formation. Avoid breathing dust.</li>
	<li>To terminate the reaction, submerge the arrays in clean tap water and wash extensively.</li>
	<li>Place the arrays between blotting paper overnight at ambient temperature to dry.</li>
	<li>Discard the arrays with obvious defects and repeat the probing protocol in such instances (Figure 4).</li>
</ol>
7. Analysis and quantification
<ol>
	<li>Scan the developed arrays at a 2,400 dots per inch (dpi) resolution using a desktop scanner. Convert the images to TIFF files and then to negatives.</li>
	<li>Using microarray analysis software (see Table of Materials), overlay the unique .gal grid file onto each microarray image to calculate the color intensity of the spot produced at each antigen binding site and subtract the local background.</li>
	<li>Export the grid data as a .txt file. These can then be manually imported into an Excel sheet for analysis.</li>
	<li>Generate a mean spot signal intensity value for each sample by averaging the spot intensity firstly across each sample dilution and then across any biological replicates included.</li>
	<li>Assign a value of 100 to the highest mean spot signal intensity and normalize the remaining data accordingly. 
	NOTE: Normalized mean spot signal intensities can then be presented as a heatmap of relative glycan epitope abundance using the conditional formatting function in Excel33, or using the geom_tile function in R ggplot2 package40,41.</li>
</ol>

Elucidating Glycan Structure and Occurrence Using Microarray Polymer Profiling

The authors declare no conflict of interest.

The MAPP technique described here is now a well-established method for glycan analysis. The basic principles were first described in 200711, but the technique has undergone continuous development in order to capitalize on the latest innovations in microarray technology, molecular probe development, and advances in our understanding of glycan biochemistry. In general, glycans, especially polysaccharides, are more challenging to analyze than proteins and nucleotides due to their structural complexity and heterogeneity45, as well as the fact that they cannot readily be sequenced or synthesized1. In many cases, no single technique can decipher glycan complexity conclusively; thus, MAPP is often used with other methods. This is one of the reasons why AIR preparation is usually chosen as a starting point for MAPP, since AIR is compatible with most other glycan analysis methods34, facilitating the subsequent comparison of datasets.
Due to homogenization of the sample prior to AIR preparation, some spatial information is invariably lost. However, as polysaccharides are sequentially released from samples, the presence of epitopes in the fractions obtained provides information about the molecular architecture and composition of that sample17. Thus, selecting an appropriate extraction regime is critical to the success of the method. Multiple parameters determine the suitability of the extraction method: cellular structure, time, temperature, pH, pressure, ionic strength of the solvent, and fineness of the solid particulate sample49. It is recommended that a range of increasingly more aggressive solvents is used to maximize the likelihood of successfully extracting constituent glycans and building a representative compositional picture of the sample. For most samples, CDTA, NaOH, and cellulase are sufficient to remove plant-derived storage and cell wall polysaccharides33,50,51,52. For some tissue samples, a hybrid extraction regime that also includes CaCl2, HCl, and Na2CO3 has been shown to be successful53, while marine microalgal samples may require the addition of ethylenediaminetetraacetic acid (EDTA)10.
Microarrays should include a range of pure, defined glycan standards to be used as positive controls5. Included standards should be modified according to the nature of the sample. Once printed, appropriate GRMPs need to be selected. The generation of hybridoma mAbs to polysaccharide structures is challenging54; glycan-binding antibodies are difficult to raise and can have low affinity55. Fortunately, gene sequence information for CBMs can be obtained with relative ease for recombinant expression4 and engineering their binding specificities56,57. Whilst an impressive catalog of GRMPs has been developed, with most now available from commercial sources, relative to the diversity of glycan structures existing in nature, only a small proportion have been produced and successfully characterised58. This can limit the ability to detect and discriminate between certain structures. It is advisable to perform an initial probing experiment using one or two probes representative of each major glycan structure anticipated to be present, for which the binding specificity is well characterized. In subsequent probing experiments, the probe list can be expanded to cover a broader range of glycans and delve deeper into fine structures.
Although mundane, ensuring that microarrays are washed thoroughly after each incubation step is fundamental to the success of the probing procedure. The ineffective removal of non-specifically bound probes is likely to obscure the result by causing a high background signal following color development. In this case, it is necessary to repeat the probing procedure, starting with a new microarray. Furthermore, arrays should be touched sparingly and only by holding the edges with forceps; the nitrocellulose membrane is brittle and easily damaged. The color development solution collects in cracks and creases, causing oversaturation, which impedes array analysis.
MAPP is quick, adaptable, and convenient. This method is compatible with animal, microbial, or plant glycans derived from any biological or industrial system, so long as they can be extracted and immobilized onto nitrocellulose, and for which one has appropriate molecular probes. The data generated provide detailed, semi-quantitative, compositional insight, which cannot readily be obtained via other glycan analysis methods.

Glycans are ubiquitous in all domains of life and exhibit unparalleled diversity in structure and function compared to other macromolecules1. However, due to their complexity, variability in biosynthesis and glycosidic linkages, and the paucity of appropriate methods for dissecting glycan structures, our understanding of this diversity in structures and functions is relatively limited2.
Many glycan analysis techniques are destructive and necessitate the breakdown of glycans into their constituent monosaccharides, which can obscure relevant three-dimensional and biological contexts3. Conversely, monoclonal antibodies (mAbs), carbohydrate binding modules (CBMs), lectins, viral agglutinins, and microbial adhesins, known collectively as glycan-recognizing molecular probes (GRMPs)4, recognize and bind to specific epitopes and can be used as tools to detect and discriminate between glycans within complex multi-glycan matrices5,6.
Here, we present microarray polymer profiling (MAPP), a rapid, versatile, and nondestructive method for glycan analysis that is applicable to a broad spectrum of biological samples. The method aims to provide a robust and high-throughput technology for analyzing glycans from diverse biological and industrial/commercial systems. MAPP unites the recognition specificity of glycan-directed molecular probes with reproducible, high-performance microarray screening technology to allow thousands of molecular interactions to be profiled in parallel. The output of this approach is diagnostic insight into the composition and relative abundance of glycans within a sample or tissue of interest.
MAPP can be used as an independent, stand-alone method, or in conjunction with other biochemical techniques, such as immunofluorescence microscopy7,8,9 and monosaccharide or linkage analysis10,11. The technique can also be used to map epitope specificities of novel GRMPs, using arrays printed with pure and structurally well-defined oligosaccharide standards12. A major advantage of MAPP over other methods, such as enzyme-linked immunosorbent assay (ELISA), is its compatibility with small sample volumes13,14. Moreover, MAPP offers significantly higher-throughput analysis15 and provides an effective form of sample preservation, as printed samples are dry and stable when immobilized onto nitrocellulose16.
The binding of GRMPs is generally dependent on the presence of a number of contiguous sugar residues that collectively form a binding site (epitope) that is unique to a particular polysaccharide class (xylan, mannan, xyloglucan, etc.)17. In contrast, the individual sugar residues (xylose, mannose, glucose) that are quantified using most biochemical techniques, for example monosaccharide composition or methylation analysis, can be components of multiple polysaccharide classes and thus difficult to assign18.
MAPP has been developed in response to a technology gap, namely the ability to rapidly analyze multiple glycans from a variety of sources using small amounts of material. MAPP capitalizes on the extensive repertoire of GRMPs that have been developed and characterized over the last three decades12,19,20,21,22,23,24,25,26,27,28,29,30,31,32. The development of MAPP has been an iterative process, with the technique being steadily refined and optimized. There is now a substantial body of literature describing the application of MAPP to various natural and industrial systems where glycans play central roles 5,6,9,10,21,33,34,35,36,37,38,39. Here, we describe the current state of the art for MAPP.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,3:1,4-&#946;-D-Glucan, Lichenan (icelandic moss)</td>
			<td>Megazyme</td>
			<td>P-LICHN</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-&#946;-D-Mannan</td>
			<td>Megazyme</td>
			<td>P-MANCB</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well microtiter plate</td>
			<td>Greiner Bio-One</td>
			<td>M1686</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl-phosphate (BCIP)</td>
			<td>Melford</td>
			<td>B74100-1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>270725</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase AffiniPure Goat Anti-Rat IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>112-055-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase AffiniPure Rabbit Anti-His Tag</td>
			<td>Jackson ImmunoResearch</td>
			<td>300-055-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Arabinoxylan (wheat)</td>
			<td>Megazyme</td>
			<td>P-WAXYL</td>
			<td></td>
		</tr>
		<tr>
			<td>Array-Pro Analyzer Software</td>
			<td>Media Cybernetics</td>
			<td>Version 6.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacillus sp. Cellulase 5A (BCel5A)</td>
			<td>NZYTech</td>
			<td>CZ0564</td>
			<td></td>
		</tr>
		<tr>
			<td>BAM antibodies</td>
			<td>SeaProbes</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Black drawing ink (indian ink)</td>
			<td>Winsor &#38; Newton</td>
			<td>GWD030</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbohydrate binding modules</td>
			<td>NZYTech</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>CCRC antibodies</td>
			<td>CarboSource</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>CDTA</td>
			<td>Sigma</td>
			<td>319945</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>PHR1552</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>1.11727</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactan (potato)</td>
			<td>Megazyme</td>
			<td>P-GALPOT</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactomannan (carob)</td>
			<td>Megazyme</td>
			<td>P-GALML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol solution</td>
			<td>Sigma</td>
			<td>49781-5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Gum tragacanth (legumes)</td>
			<td>Sigma-Aldrich</td>
			<td>G1128</td>
			<td></td>
		</tr>
		<tr>
			<td>INCh antibodies</td>
			<td>INRA</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>LM and JIM antibodies</td>
			<td>PlantProbes</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Marathon Argus Microarray Printer</td>
			<td>ArrayJet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma&#160;</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibodies</td>
			<td>Biosupplies Australia</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Sigma</td>
			<td>452882</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitro-blue tetrazolium (NBT)</td>
			<td>Melford</td>
			<td>N66000-1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Thermo Fisher Scientific</td>
			<td>88018</td>
			<td></td>
		</tr>
		<tr>
			<td>Pectin (degree of methyl esterification 46%)</td>
			<td>Danisco</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ProClin 200</td>
			<td>Sigma</td>
			<td>48171-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhamnogalacturonan (soybean pectic fibre)</td>
			<td>Megazyme</td>
			<td>P-RHAGN</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating mixer</td>
			<td>Fisher Scientific</td>
			<td>88-861-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating/rocking Shaker</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skimmed milk powder</td>
			<td>Marvel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin filter</td>
			<td>Costar Spin-X</td>
			<td>8160</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel beads</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>93362</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P9416-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylan (beechwood)</td>
			<td>Megazyme</td>
			<td>P-XYLNBE</td>
			<td></td>
		</tr>
		<tr>
			<td>Xyloglucan (tamarind)</td>
			<td>Megazyme</td>
			<td>P-XYGLN</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Glucan (oat)</td>
			<td>Megazyme</td>
			<td>P-BGOM</td>
			<td></td>
		</tr>
	</tbody>
</table>

microarray polymer profiling (mapp) for high-throughput glycan analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

MAPP was applied to determine the glycan composition of agricultural biomass waste, comprising mango peels from several northern Thai varieties, Coffea arabica cherry pulp and coffee bean processing waste, and root, stem, and leaf tissue from Thai black garlic, Allium sativum var. ophioscorodon. Several plant-derived polysaccharides are used in the food industry as functional ingredients42,43. Thus, the aim of this experiment was to deduce whether these abundant and currently underutilized agro-industrial waste materials may provide a source of value-added pure polysaccharides.
AIR material is routinely employed to prepare samples intended for glycan analysis44. There are several advantages to using AIR; treatment with solvents effectively removes endogenous CAZymes, metabolites, small saccharides, lipids, and pigments, resulting in samples enriched with polysaccharides and structural proteins34. Furthermore, producing AIR is a fast and effective way to increase sample longevity, as it is thermostable and can be stored for several years.
Three mixed fractions of constituent glycans were sequentially extracted from plant AIR material using CDTA, NaOH, and cellulase. CDTA chelates Ca2+ ions, which allow the removal of Ca2+ crosslinked de-esterified pectins from plant cell walls45. Alkaline conditions allow predominantly hemicelluloses, such as mannan, xylan, and &#946;-glucan, to be released due to the disruption of hydrogen bonding and saponification of ester linkages between cellulose microfibrils and hemicellulose, and lignin and hemicellulose, respectively46. A recombinant endo-1,4-&#946;-glucanase from Bacillus spp. was used to degrade amorphous regions of the structural cellulose microfibrils, releasing residual glycans bound to cellulose within the cell walls47. Although this method effectively separates glycans into these three broad groups, it should be noted that the samples are not pure; by the very nature of the extraction method, hemicellulose, if present in the sample, will inevitably be extracted and subsequently detected to varying degrees in the CDTA and cellulase fractions. Likewise, some pectin will be detected in the NaOH extraction if present in the sample.
A non-contact, piezoelectric microarray printing robot was used to immobilize extracted glycan fractions onto nitrocellulose via non-covalent attachment11, forming 300 identical microarrays. Defined glycan standards (Table 1) were also included in the printed microarrays as positive controls (Figure 5). The MAPP binding profile obtained for the selected glycan standards corresponds to previously reported epitope specificities. For example, LM21 exhibited strong binding to multiple mannan polysaccharides (galactomannan and glucomannan), while LM22 exhibited only weak binding to galactomannan25. Similarly, LM19 preferentially bound to de-esterified homogalacturonan48&#160;and LM15 bound to tamarind seed xyloglucan23.
The relative abundance of 16 epitopes, diagnostic of non-cellulosic plant cell wall polysaccharides, were detected by&#160;the attachment of&#160;glycan-directed monoclonal antibodies (Table 2) to printed extracts (Figure 6). The majority of extracted glycans were detected within the alkaline NaOH fraction. Strong binding signals were recorded for mAbs LM10 and LM11, representing xylan/arabinoxylan, within the peels of all the mango varieties tested. Within the garlic samples, LM10 and LM11 bound preferentially to root tissue extract (Garlic R) and exhibited only weak binding to the leaf tissue extract (Garlic L). LM19, representing partially methyl-esterified or un-esterified homogalacturonan, bound strongly to some mango variety extracts (Aokrong and Talabnak), but bound only weakly, or its binding was undetectable, in other varieties (Chokanan, Mamkamdang, Mahachanok, and Nga). In addition, LM19 bound only to the coffee pulp fractions and did not bind to the coffee bean processing waste material, previously thought to be composed of semi-purified coffee pectin (unpublished data).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65443/65443fig01.jpg" /> 
Figure 1: Major experimental steps in the MAPP method. (A) Samples are homogenized to form fine powders. (B) The homogenized samples are processed to isolate their AIRs. (C) The constituent glycans are sequentially extracted using a tailored extraction regime. (D) The extracted glycan fractions, ink, and GSB are transferred into 384-well plates, according to the plate layout, for printing onto nitrocellulose. (E) The printed microarrays are probed with selected GRMPs. (F) GRMP binding to the printed glycan fractions is quantified and analyzed before data are presented as a heatmap. <a href="https://www.jove.com/files/ftp_upload/65443/65443fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65443/65443fig02.jpg" /> 
Figure 2: Example of a 384-well plate layout for sample, ink, and GSB loading with four dilutions per extracted glycan sample/standard. Different colors denote samples arising from different extraction reagents, while different shades represent serial dilutions. The first number in the code represents the sample number, while the end number represents the dilution number (D1 denotes dilution one, D2 denotes dilution two, and so on). For example, a well labeled &#39;12D3&#39; represents glycan sample 12, dilution three. Well plates should be divided into eight identical sections comprising six columns and eight rows. The first section of the first plate should contain only ink and buffer and resemble the example plate layout. Extracted glycan samples can then be loaded into subsequent plate sections according to the plate layout. Different extraction reagents should not be loaded into the same plate section. If there are insufficient samples to fill an entire section, fill all remaining wells in that section with buffer; do not leave any wells empty. If multiple plates are required, the next section after all samples have been loaded should contain three alternating columns of ink and GSB-this may not be section eight, depending on the number of samples being printed. <a href="https://www.jove.com/files/ftp_upload/65443/65443fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65443/65443fig03.jpg" /> 
Figure 3: Schematic representation of printed microarray design. <a href="https://www.jove.com/files/ftp_upload/65443/65443fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65443/65443fig04.jpg" /> 
Figure 4: Representative microarrays. (A) No binding. (B) The binding signal is obscured by high background signal. (C) Generalized blue/purple staining due to oversaturation with NBT/BCIP. (D) Defective probing due to high substrate concentration. (E) Defective printing due to the unclean print head. (F) Strong binding to few samples. (G) Strong binding to many samples. <a href="https://www.jove.com/files/ftp_upload/65443/65443fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65443/65443fig05.jpg" /> 
Figure 5: Monoclonal antibody binding to defined glycan standards, included to validate the printing and probing process. <a href="https://www.jove.com/files/ftp_upload/65443/65443fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65443/65443fig06.jpg" /> 
Figure 6: MAPP of glycans extracted from agricultural biomass waste. The samples include coffee pulp waste (coffee pulp and coffee pectin), mango peels from several Thai varieties (AO, Aokrong; KO, Kam; RD, Rad; CH, Chokanan; MA, Mamkamdang; TL, Talabnak; MH, Mahachanok; NG, Nga) and black garlic leaves (Garlic L), stem (Garlic S), bulb (Garlic BG), and roots (Garlic R), using CDTA, NaOH, and cellulase (Bacillus spp. cellulase 5A). <a href="https://www.jove.com/files/ftp_upload/65443/65443fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Defined commercial polysaccharide standards used in MAPP analysis as positive controls. <a href="https://www.jove.com/files/ftp_upload/65443/65443_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Glycan-directed monoclonal antibodies selected for the interrogation of extracted plant glycan microarrays. <a href="https://www.jove.com/files/ftp_upload/65443/65443_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about MAPP Protocol â€“ Advancing Glycan Analysis at JoVE.com

Author Spotlight: MAPP Protocol &#8211; Advancing Glycan Analysis 

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...

microarray-polymer-profiling-mapp-for-high-throughput-glycan-analysis

Universidad San Sebastian

Research

JoVE Journal

Biology

1.6K Views.  Newcastle University. Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples. 

Video: Author Spotlight: MAPP Protocol – Advancing Glycan Analysis 

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...