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SuperEpoxy and SuperEpoxy 2 Protein

ArrayIt® is pleased to announce SuperEpoxy 2, the newest version of our original SuperEpoxy Microarray Substrates product line. ArrayIt® substrates are the only microarray substrates in the world that offer a polished atomically smooth glass surface (±20 angstroms), for the ultimate in silicon dioxide homogeneity and data precision. SuperEpoxy 2 Substrates are manufactured to “open platform” dimensions (25 x 76 mm), cleaned at the atomic level in state-of-the-art class 100 cleanrooms, and treated with our ultra-pure epoxy surface chemistry for highly-reactive covalent coupling efficiency and low background. Heat-sealing in anti-static packaging improves shelf life and eliminates electrostatic accumulation. SuperEpoxy 2 Substrates have a proprietary corner chamfer for unambiguous side and end orientation, which greatly improves usability during manufacturing. SuperEpoxy 2 products outperform standard optical quality glass from other vendors and are the substrates of choice for high-density protein microarray manufacturing applications using contact and non-contact printing methods. SuperEpoxy 2 products are compatible with all major brands of microarrayers and scanners.

Table of Contents

  • Introduction
  • Quality Control
  • Product Description
  • Technical Note
  • Technical Assistance
  • Short Protocols
  • Complete Protocols
  • Recommended Equipment
  • Troubleshooting Tips
  • Ordering Information
  • Storage Conditions
  • Warranty

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your proteomics research. This booklet contains a complete set of protocols outlining the steps and principles needed to use ArrayIt® SuperEpoxy Substrates.

Quality Control
Arrayit assures the performance of this product. The finest scientific research went into the development of this product. Rigorous quality control monitoring on a lot-by-lot basis guarantees that the product exceeds the highest industry standards.

Product Description
Arrayit's ArrayIt® SuperEpoxy Microarray Substrates provide the highest quality glass microarray printing substrate at an affordable price. All of our substrates are manufactured in state-of-the art class 10 cleanrooms, with 0.1 µm filtered air, and temperature and humidity control. Cleanroom manufacture eliminates contamination of the microarray surface with particulates, proteases, nucleases and other contaminants that impair the quality of microarray experimentation. Compare our SuperEpoxy Substrates to traditional microarray slides and you will see the difference.

Users will appreciate the following product features:

  • Polished to atomic surface smoothness <±20 angstroms over 1.0 µm2
  • Superior substrate flatness of <0.036 mm over 25 x 76 mm
  • Only polished glass substrate/slide in the microarray industry
  • Homogenous SiO2 groups provide superior silane reactivity
  • Vastly superior to unpolished or flame polished glass from other vendors
  • Ultimate surface for ultra-high density DNA and protein microarray manufacturing including micro-mirror, photolithography and contact printing
  • Used by high-volume microarray manufacturing companies
  • Used by overseas companies for diagnostics
  • Topological smoothness ensures uniform hybridization layer and scanning
  • Manufactured in a state-of-the-art class 100 cleanrooms
  • Ultra-low intrinsic fluorescence and background noise
  • Can be used as a starting point for gel and membrane coatings (e.g. polyacrylamide and nitrocellulose)
  • Used by major academic core facilities
  • Open platform dimensions compatible with all major brands of microarrayers and scanners
  • Precise physical dimensions (25 ± 0.2 mm x 76 ± 0.3 mm x 0.940 mm ± 0.025 mm)
  • Proprietary corner chamfer (1.4 mm) provides unambiguous side and end orientation to simplify printing and processing
  • Finished edges enhance user safety
  • Excellent refractive index, transmission and hardness specifications
  • Sophisticated anti-static packaging improves usability
  • Offered with or without bar-codes
  • Custom laser and chrome fiducials available upon request
  • Product arrives “ready to print” with no additional processing required
  • High-volume 100,000 piece per month manufacturing capabilities
  • Free of particulate, protease and nuclease contamination
  • High efficiency covalent coupling via highly reactive epoxide groups
  • Protein coupling via surface lysine and arginine residues
  • Supports both contact printing and ink-jet printing
  • Protein coupling complete within minutes after printing
  • No cross-linking or baking required for coupling
  • “Zero” background fluorescence
  • Optimal reactive group density of 5 x 1012 reactive groups per mm2
  • Uniform epoxide density of ±5% across the substrate
  • Print proteins, enzymes, antibodies, receptors, antigens, and peptides
  • Print pure proteins, recombinant proteins and cellular extracts
  • Uniform feature size
  • 25 substrates per box
  • Anti-static and impact resistant packaging
  • Three month shelf life at room temperature

Technical Note
It may be necessary to add protease or phosphatase inhibitors to the proteins mixed with Protein Printing Buffer printed on SuperEpoxy Substrates to increase the stability of certain proteins or protein extracts. Protein stability is greatly enhanced by the use of Protein Printing Buffer. The stability of the source plate of samples can be further enhanced if microarray manufacturing is performed at 4°C using the SpotBot 2 Protein Edition Personal Microarrayer or the NanoPrint Protein Edition Microarrayer.

Figure 1. Protein microarrays. (A) Proteins bind to epoxide groups on the SuperEpoxy surface. Primary amines (lysine shown) on the protein surface act as nucleophiles, attacking epoxy groups and coupling the protein covalently to the surface. (B) Schematic illustration of the antibodies shown in panel C. The yellow icons depict printed target antibodies attached to the substrate, and the green and red icons depict Cy3- and Cy5-labelled secondary antibodies, respectively. (C) Ten monoclonal and polyclonal antibody samples from QED Biosciences, Inc. (San Diego, CA) were suspended at a final concentration of 0.1-0.5 µg/µl in 1X ArrayIt® Protein Printing Buffer, and printed five times each on ArrayIt® SuperEpoxy Substrates using a SpotBot Protein Edition Personal Microarrayer running SMP9 Micro Spotting Pins.  Printed microarrays were blocked for 30 min with 1X phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), and washed three times for 2 min each in 1X PBS.  The antibody microarray was incubated for 60 min with a fluorescent sample containing 1X PBS, 0.5% BSA, and 1:1,000 dilutions of Cy3-goat anti-rabbit and Cy5-goat anti-mouse antibodies.  The microarray was washed 3 times for 5 min each with 1X PBS and scanned with a ChipReader microarray scanner from Virtek Vision (Ontario, Canada) set at 100 Laser power, 1000 PMT, and Gain 10 in the Cy3 and Cy5 channels.  A two-color composite image was analyzed using Quantarray software from Perkin Elmer (Boston, MA).  All incubations and washes were performed at room temperature (22°C).  The data reveal the coupling efficiency of the antibodies to the SuperEpoxy surface, and the efficiency and specificity of the antibody-antibody binding reactions. Reprinted courtesy of Nature Medicine (Stears et al., Nat. Med. 9, 140-145, 2003).

Figure 2. SuperEpoxy is a perfect surface for whole proteome microarrays and screening expressed protein libraries in the microarray format.  Libraries of express proteins are easily printed into microarrays with the Stealth Micro Spotting Device.  For detection of the printed proteins, samples are labeled directly with fluorescence.  A variety of protein, protein interactions can be implemented including analyzing antibody specificity.

  

Figure 3. The SuperEpoxy substrate is suitable for immobilizing both antigens and antibodies for implementing M-ELISA (micro enzyme linked immunosorbent assay) and antibody microarrays.  Detection can be accomplished with secondary antibodies labeled with AP, HRP, biotinylated secondary antibodies that bind labeled streptavidin or direct labeling.  A variety of sandwich assays can be accomplished.

Figure 4. SuperEpoxy can be used to implement reverse phase microarrays. These microarrays are used to profile bound complex cell lysates, cells captured by LCM or serum samples printed into microarrays. The bound samples can be probed with antibodies for the detection of antigens present in the printed samples. Detection is accomplished with a secondary antibody.

Figure 5. SuperEpoxy can immobilize synthetic proteins, peptides and engineered proteins to detect the presence of proteins in complex samples. Unique protein binding events or protein/protein interactions can be detected.

Figure 6.  Reactive epoxide surface chemistry binds proteins covalently in several different ways and, therefore, a high number of protein molecules can be captured on the surface for various binding reactions. Proteins bind to the surface in a spatially random manner, which maintains the accessibility of all protein epitopes in each printed microarray spot. Molecular linkers between the glass substrate slide and the epoxide groups facilitate interactions between bound protein molecules and their binding partners in solution.

Figure 7. Correct Substrate Orientation. Shown is a graphic of two ArrayIt® Microarray Substrates, showing the correct and incorrect orientation for use. In the correct orientation (blue graphic), the chamfer will be located in the upper right corner and samples should be printed on the side facing upward, which is the same side that contains the word “Correct!”. In the incorrect orientation (red graphic), the chamfer will be located in the upper left corner, placing the backside facing upward, which is the side that contains the word  “Incorrect!”. Only one side of ArrayIt® Microarray Substrates is suitable for printing. Please print on the correct side only.

Figure 8. Nine pAb's with different Ag specificities were spotted in triplicate onto SuperEpoxy Microarray Substrates using 946MP3 Micro Spotting Pins in a stabilizing protein printing buffer and stored for 2 months at room temperature in the dark. These were assembled in a 96-well microtiter plate format such that a complete set of antibodies is available for multiplex assays per well. Mixtures of biotinylated Ag's were added to each well and allowed to bind to their specific pAbs. The level of specifically bound Ag was quantified by using a streptavidin-Cy3 secondary antibody. In cases where no specific Ag was added for a particular Ab there is no signal detected.

Figure 9. SuperEpoxy 2 substrates are also available in the SuperMask format.  Custom masks are available. Click here for more information.

Protein Short Protocol (Steps 1-7)
1. Suspend protein samples in Protein Printing Buffer at 0.1-0.5 µg/µl.
2. Print protein samples onto SuperEpoxy Substrates.
3. Block with BlockIt and process the printed protein microarrays.
4. React the processed microarrays with fluorescent samples.
5. Wash the microarrays to remove un-reacted fluorescent material.
6. Scan the microarray to produce a fluorescent image.
7. Quantitate and model the fluorescent data.

Protein Complete Protocol (Steps 1-7)
1. Suspend protein samples in Protein Printing Buffer.  Obtain 0.2-1.0 µg/µl protein samples in 1X phosphate buffered saline (PBS) and add an equal volume of 2X Protein Printing Buffer.  Mix the samples by pipetting up and down 10 times.  Protein samples should be free of aggregates and particulates that can clog printing devices and impair attachment to the microarray substrate.  Aggregates and particulates can be removed by centrifugation, dialysis and/or filtration.  A 50kD protein at 1 µg/µl concentration has a concentration of 20 µM.  At 30% coupling efficiency, a 20 µM protein will produce a target density of 1011 proteins per mm2 of substrate. Certain proteins or protein extracts are more stable at 4°C.  Keeping the protein samples cool may improve protein stability. Stability can also be improved in some cases by the addition of protease and phosphatase inhibitors, or by the use of a SpotBot Protein Edition Personal Microarrayer or NanoPrint Protein Edition Enterprise Level Microarrayer equipped with a cooled platen or plate cooling apparatus. Make sure protein samples are mixed thoroughly before printing.

2. Print protein samples onto SuperEpoxy Substrates. The SuperEpoxy surface couples proteins extremely efficiently owing to the high reactivity of the epoxide groups (see Fig. 2).  Coupling is complete shortly after printing, and proteins retain protein-protein binding and enzymatic activity (see Fig. 1) but it is good protocol to let microarrays sit overnight prior to processing.. Printed microarrays should be stored unprocessed to protect coupled molecules. Protein Printing Buffer contains components that stabilize printed proteins. Processing should be performed just prior to use for best performance.

3. Block and process the printed protein microarrays.  Prior to using the microarray, wash the printed microarrays to remove unbound protein molecules and buffer components from the SuperEpoxy Substrates. Protein binding to the SuperEpoxy surface is extremely stable and the microarrays can be washed, blocked and reacted without sufficient loss of coupled protein.  For best results block the surface using 1X Blocking Buffer. Satisfactory results can be obtained with a 60-minute incubation at room temperature in a buffer containing 1X PBS + 1% bovine serum albumin (BSA). A High Throughput Wash Station or an equivalent device can be used for washes. Blocking can be performed with very gentle buffer agitation and the stir plate set on a low speed or under a coverslip. The blocking step will couple reactants to the open epoxide groups and prevent background fluorescence. After blocking, wash the microarrays to remove the excess blocking buffer. Washing three times for 2 min each at room temperature with 1X PBS in a High Throughput Wash Station works well.

4. React the processed microarrays with fluorescent samples. Processed microarrays containing coupled target proteins can be reacted with fluorescent samples to accomplish a variety of binding assays (see figures 1-5). Binding reactions can be performed in 1X BlockIt buffer + fluorescently labeled proteins diluted 1:1,000 or labeled cellular extracts. Alternatively PBS + 0.5% BSA can be used in cases when BlockIt is not available. Fluorescent samples can be incubated as a droplet on the printed microarray, underneath a cover slip, or in a micro-fluidics chamber.  A 60-minute incubation at room temperature is usually sufficient to obtain strong binding and intense fluorescent signals (see Fig. 1). Reactions can be performed at 37 degrees C or at room temperature. A Hybridization Cassette can be used to prevent sample evaporation during prolonged (1-12 hour) binding reactions.  Multiplexed reaction cassettes are available.

5. Wash the microarrays to remove un-reacted fluorescent material. Once the binding reaction between the bound target proteins and the fluorescent protein probe molecules is complete, wash the microarray to remove the unbound fluorescent material. Washes can be performed three times for 5 min each at room temperature in 1X PBS in a High Throughput Wash Station or equivalent device. After the wash procedure, excess buffer should be removed from the surface by tapping the substrate on a lint-free cloth or by centrifugation with a Microarray High-Speed Centrifuge.

6. Scan the microarray to produce a fluorescent image. The fluorescent protein microarray can be scanned or imaging using any of a number of high quality commercial detection instruments from ArrayIt®, Perkin Elmer, BioRad, Axon, API, Tecan, and many others. Instrument settings can be adjusted to optimize the imagine process.

7. Quantitate and model the fluorescent data using Mapix or other quantification software program.  Protein microarray data from the fluorescent image can be quantified, mined and modeled using many different commercial software packages.

Storage
When using SuperEpoxy and SuperEpoxy 2, it is best to use the product within 2 months of the manufacturing date. For example, lot 050625, was made on June 25, 2005. Once microarrays are printed, samples are covalently bound and stable for long periods of time. Protein microarrays scan be stored at 4C or at room temperature. DNA microarray storage conditions are clean and dry. These substrates are made often and customers are always guaranteed a fresh lot. SME and SME2 can also be made to order and made on custom size substrates and wafers, Contact
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Troubleshooting Tips
Poor printing quality:

  • Incomplete mixing of protein samples
  • Poor printing environment (50% humidity and 25°C recommended).
  • Clogged Micro Spotting Pins
  • For more information on troubleshooting printing tips please click here.

Poor protein coupling:

  • Contaminants in samples
  • Poor printing buffer (PPB highly recommended)
  • Weak fluorescent signals:

Poor binding of fluorescent proteins

  • Probe labeling inefficient
  • Washes too harsh

Recommended Equipment and Reagents
NanoPrint™ Microarrayers
SpotBot® 2 Personal Microarrayers
SpotLight™ Microarray Scanners
Microarray Hybridization Cassettes
High Throughput Wash Stations
Microarray High-Speed Centrifuges
Protein Printing Buffer
BlockIt™ Blocking Buffer
Microarray Air Jet
Microarray Cleanroom Wipes
Green540 and Red640 Reactive Fluorescent Dyes

Ordering Information

Product

Description

Catalog ID

SuperEpoxy 2

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity and extremely low background fluorescence, 25 x 76 mm, 25 pieces per box.

SME2

SuperEpoxy 2 Barcode

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity and extremely low background fluorescence, 25 x 76 mm and barcode, 25 pieces per box.

SME2BC

SuperEpoxy 2 (Box of 5)

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity and extremely low background fluorescence, 25 x 76 mm, 5 pieces per box.

SME2F

SuperEpoxy 2 Barcode (Box of 5)

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity and extremely low background fluorescence, 25 x 76 mm and barcode, 5 pieces per box.

SME2FBC

MirrorEpoxy 2

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity, extremely low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm, 25 pieces per box.

MRE2

MirrorEpoxy 2 Barcode

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity, extremely low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm and barcode, 25 pieces per box.

MRE2BC

MirrorEpoxy 2 (Box of 5)

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity, extremely low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm, 5 pieces per box.

MRE2F

MirrorEpoxy 2 Barcode (Box of 5)

Most advanced epoxy microarray substrate slide on the market, polished atomically flat glass, highly-reactive epoxy surface chemistry, ultra-high covalent binding capacity, extremely low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm and barcode, 5 pieces per box.

MRE2FBC

SuperEpoxy

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, 25 x 76 mm, 25 pieces per box.

SME

SuperEpoxy Barcode

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, 25 x 76 mm and barcode, 25 pieces per box.

SMEBC

SuperEpoxy (Box of 5)

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, 25 x 76 mm, 5 pieces per box.

SME5

SuperEpoxy Barcode (Box of 5)

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, 25 x 76 mm and barcode, 5 pieces per box.

SME5BC

MirrorEpoxy

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm, 25 pieces per box.

MRE

MirrorEpoxy Barcode

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm and barcode, 25 pieces per box.

MREBC

MirrorEpoxy (Box of 5)

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm, 5 pieces per box.

MRE5

MirrorEpoxy Barcode (Box of 5)

Advanced epoxy microarray substrate slide, polished atomically flat glass, reactive epoxy surface chemistry, high covalent binding capacity, low background fluorescence, reflective backside coating for 2-10X stronger signals, 25 x 76 mm and barcode, 5 pieces per box.

MREBC5

Storage Conditions
Arrayit's SuperEpoxy Microarray Substrates are manufactured and packaged in state-of-the-art class 100 cleanroom facilities and are stable at room temperature (20-25°C) for 3 months from the date of receipt. Substrates should be printed within the 3 month period for optimal protein and DNA coupling efficiency. SuperEpoxy Substrates printed after the 3 month storage period will exhibit a gradual reduction in coupling efficiency. Prior to use, store in the vacuum-sealed shipping envelope and avoid exposure to elevated temperature.

Warranty
ArrayIt® brand products have been scientifically developed and are sold for research purposes. Extreme care and exact attention should be practiced in the use of the materials described herein. All ArrayIt® Brand products are subject to extensive quality control and are guaranteed to perform as described when used properly. Any problems with any ArrayIt® Brand product should be reported to Arrayit immediately. Arrayit’s liability is limited to the replacement of the product, or a full refund. Any misuse of this product including the use of the wrong protocols is the full responsibility of the user, and Arrayit makes no warranty or guarantee under such circumstances.