Construction and Use of Glycan Microarrays

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Glycosylation is an important post?translational modification that influences many biological processes critical for development, normal physiologic function, and diseases. Unfortunately, progress toward understanding the roles of glycans in biology has been slow due to the challenges of studying glycans and the proteins that interact with them. Glycan microarrays provide a high?throughput approach for the rapid analysis of carbohydrate?macromolecule interactions. Protocols detailed here are intended to help laboratories with basic familiarity of DNA or protein microarrays to begin printing and performing assays using glycan microarrays. Basic and advanced data processing are also detailed, along with strategies for improving reproducibility of data collected with glycan arrays. Curr. Protoc. Chem Biol. 2:37?53. © 2010 by John Wiley & Sons, Inc.

Keywords: glycosylation; microarray; neoglycoconjugate; carbohydrate?dependent binding; serum antibody profile

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Introduction
Basic Protocol 1: Production of Glycan Microarrays
Basic Protocol 2: Glycan Array Profiling of Carbohydrate‐Binding Properties
Basic Protocol 3: Scanning and Data Analysis of Glycan Microarrays
Alternate Protocol 1: Advanced Processing and Methods: Extending the Dynamic Range of Pixel Intensity Measurements
Alternate Protocol 2: Normalize Slide Using Reference Sample
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Production of Glycan Microarrays

Materials

Neoglycoproteins and/or glycoproteins (see protocol introduction above regarding sources)
Printing buffer (see recipe )
Controls: unmodified BSA and HSA (negative controls) and BSA conjugated with Cy3 or Alexa Fluor 647

384‐well V‐bottom sample plates with lids (X6004, Genetix)
Aluminum plate seals (07‐200‐683, Costar)
Centrifuge with microtiter plate carrier
Robotic Microarray Printer (MicroGrid II, Genomic Solutions; http://bioinformatics.genomicsolutions.com/)
Microscope (dissecting or basic optical transmission microscope)
Pins (Stealth Microspotting SMP3 Pins, Arrayit; http://www.arrayit.com/)
Epoxide Coated Slides (SuperEpoxy 2 Premium Microarray Substrates, Arrayit; http://www.arrayit.com/)
Hygrometer

Basic Protocol 2: Glycan Array Profiling of Carbohydrate‐Binding Properties

Materials

Glycan arrays ( protocol 1 )
Blocking buffer: 3% (w/v) bovine serum albumin (BSA, Ig‐free; Sigma, cat. no. A‐3059) in 1× phosphate‐buffered saline (PBS; see recipe for 10×)
PBST array wash buffer (see recipe )
Appropriate incubation buffer for sample, consisting of BSA and PBST (Table 9.2.2800 )
Sample (see Table 9.2.2800 for appropriate concentrations)
Reference sample, e.g., pooled serum or biotinylated lectins known to bind glycans on the array
Secondary antibody and/or Cy3‐conjugated streptavidin (Jackson ImmunoResearch) and appropriate buffer (Table 9.2.2800 )

Microscope (dissecting or basic optical transmission microscope)
Slide module (ProPlate, Invitrogen)
Adhesive seals for slide module (ProPlate, Invitrogen)
Orbital shaker
50‐ml conical centrifuge tubes

Centrifuge

Table 9.2.2 MaterialsRecommended Buffers and Starting Dilutions of Samples for Incubation on Glycan MicroarrayRecommended Secondary Reagents and Conditions

Sample type	Incubation buffer a	Concentration	Conditions
Lectin	1% BSA in PBST	1‐50 µg/ml	Room temperature for 2 hr
Monoclonal antibody	3% BSA in PBST	1‐50 µg/ml	37°C while gently shaken for 2‐4 hr
Serum antibodies	3% BSA in PBST	1:50 to 1:200	37°C while gently shaken for 4 hr
Sample type	Secondary label	Buffer	Concentration	Conditions
Biotinylated lectin	Cy3‐Streptavidin	1% BSA in PBS	2 µg/ml	Room temperature for 2 hr
Monoclonal antibody	Monoclonal antibody specific for species and isotype of primary Ab	3% BSA in PBS	2 µg/ml	37°C while gently shaken for 2 hr
Human serum antibodies	Monoclonal anti‐human IgG, IgM, and/or IgA	3% HSA + 1% BSA in PBS	2 µg/ml	37 °C while gently shaken for 2 hr

^a PBST: PBST: see recipe for PBST array wash buffer in Reagents and Solutions.

Table 9.2.3 MaterialsRecommended Buffers and Starting Dilutions of Samples for Incubation on Glycan MicroarrayRecommended Secondary Reagents and Conditions

Sample type	Incubation buffer a	Concentration	Conditions
Lectin	1% BSA in PBST	1‐50 µg/ml	Room temperature for 2 hr
Monoclonal antibody	3% BSA in PBST	1‐50 µg/ml	37°C while gently shaken for 2‐4 hr
Serum antibodies	3% BSA in PBST	1:50 to 1:200	37°C while gently shaken for 4 hr
Sample type	Secondary label	Buffer	Concentration	Conditions
Biotinylated lectin	Cy3‐Streptavidin	1% BSA in PBS	2 µg/ml	Room temperature for 2 hr
Monoclonal antibody	Monoclonal antibody specific for species and isotype of primary Ab	3% BSA in PBS	2 µg/ml	37°C while gently shaken for 2 hr
Human serum antibodies	Monoclonal anti‐human IgG, IgM, and/or IgA	3% HSA + 1% BSA in PBS	2 µg/ml	37 °C while gently shaken for 2 hr

NOTE: Prepare all solutions fresh on the day of the experiment.NOTE: All BSA and HSA must be globulin‐free to avoid high levels of background binding. Albumin that has not been sufficiently purified contains globulins, which will interfere in the assay. Other sources of albumin purified by gel electrophoresis may be substituted after confirming that they do not contain contaminating immunoglobulins that bind to the array.

Basic Protocol 3: Scanning and Data Analysis of Glycan Microarrays

Materials

Arrays ( protocol 2 )
Fluorescence scanner (GenePix 4000A, Molecular Devices)
Image processing software (GenePix Pro 6.0, Molecular Devices)
Microsoft Excel

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Figures

Figure 1. Coordination of slide layout, configuration of sample plate, and pin positioning. (A ) An example is shown with four pins loaded in the pin tool. Pins are lowered into the wells of a source plate (e.g., 384‐well plate), allowing the pins to fill with neoglycoprotein solution. The robot then moves the pin head to the slides to print arrays of spots. 16 complete arrays are printed on each slide. The spacing of the pins and the array layout should match the spacing of the 16‐well slide module. (B ) Slides are fitted with a 16‐well module to form 16 separated wells. The wells have the same spacing as is normally found on 96‐well plates, and a multichannel pipettor can be used to add solutions to the wells. The slide shown in the picture has a mask that subdivides the slide into 16 areas; however, this is shown for illustrative purposes, and slides without masks can also be used.

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Figure 2. Inspection of pins prior to printing. Magnified views of the pins are shown. The full length of the pin should be inspected for any debris that may clog the channel. A clean pin is free of debris throughout its channel and near the pin tip.

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Figure 3. Microscope images of printed arrays. The figure shows magnified portions of arrays after printing but prior to an assay. (A ) High‐quality printing produces uniform spots evenly spaced on the glass surface, which is free of debris. (B ) Small spots may be due to a partially clogged pin or low volume of sample in the pin due to poor pin loading or excessive pre‐spotting. (C ) Debris on the surface may have no or high signal, depending on its fluorescence. (D ) Missing spots are typically due to pin sticking.

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Figure 4. Scans of processed arrays. Examples of images scanned using a fluorescence scanner. (A ) High‐quality results show circular spots of varying intensity but uniform size. (B ) Under higher magnification, high‐quality spots have homogeneous intensity throughout the spot, and duplicate spots have nearly identical intensity. Printing and processing problems can result in variations in intensity across individual spots, irregular spot morphology, or missing spots.

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Videos

Literature Cited

Literature Cited
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Construction and Use of Glycan Microarrays

Abstract

Table of Contents

Materials

Basic Protocol 1: Production of Glycan Microarrays

Basic Protocol 2: Glycan Array Profiling of Carbohydrate‐Binding Properties

Basic Protocol 3: Scanning and Data Analysis of Glycan Microarrays

Figures

Videos

Literature Cited