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Products - Microarray Substrates & Slides - 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 atomic flat 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
TeleChem 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
TeleChem'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 features of SuperEpoxy Substrates (SME):
- Manufactured in state-of-the-art class 10 microarray cleanrooms
- Free of particulate, protease and nuclease contamination
- Standard 1” x 3” size (76 x 25 x 0.96 mm)
- Precise dimensionality: 25 ± 0.2 mm x 76 ± 0.3 mm x 0.960 ± 0.025 mm
- Polished flat to 50 Ĺ (20 atoms) for superior performance
- Corner chamfer (upper right corner) for unambiguous orientation
- 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 manufacture is performed at 4°C. The SpotBot Personal Microarrayer and NanoPrint Microarrayer can be moved into a cold room easily, and operated at 4°C. A special “cold platen” is also available these systems if a 4°C printing temperature is required in an ambient laboratory environment.
The “substrate noise” is the sum of all non-sample and non-instrument contributions to the background reading including intrinsic fluorescence of the substrate and reflection off the substrate surface. Because substrate noise is measured using a scanning or imaging device, as a practical matter substrate noise typically includes intrinsic fluorescence and reflection, as well as all of the sources of instrument noise (e.g. dark current, shot noise, electronic noise and optical noise). Most modern scanners and imagers have very low instrument noise, which means that intrinsic fluorescence and reflection dominate the substrate noise reading. Substrate noise is obtained by measuring the fluorescent reading of the substrate “right out of the box” and prior to reacting the substrate with a fluorescent sample. Please do not confuse substrate noise (i.e. background before the substrate is reacted with a sample) and microarray noise (i.e. background after the substrate has been reacted with a sample), as these two readings are very different. For nearly all applications and assays, microarray noise greatly exceeds substrate noise and therefore concerns about substrate noise are entirely academic because the latter does not contribute to the total background reading observed when the product is used in real experiments (see below).
The table below summarizes the substrate noise (actually intrinsic fluorescence + reflection + instrument noise) observed with our substrates. Substrate noise readings were taken at very high sensitivity (90% laser and 90% photomultiplier tube or PMT), settings that are well beyond those used for biological experiments. Typical instrument settings for biological experiments with the ScanArray Express are 20-40% laser power and 80% PMT. The ScanArray Express has 20- to 30-fold greater sensitivity than the ScanArray 3000, and correspondingly the substrate noise readings are up to 30-fold higher on the Express compared to the 3000. Unmodified glass substrates (i.e. SuperClean) produce the lowest substrate noise readings, followed SuperEpoxy, SuperAmine, and SuperAldehyde. Organic treatments (e.g. epoxy, amine and aldehyde) increase substrate noise compared to naked glass because organic molecules formed during derivation fluoresce at an extremely low but detectable level. For nearly all applications and assays, the non-sample contributions to background noise (intrinsic fluorescence, reflection, and instrument noise) are much less than the background noise contributed when the substrate or microarray is reacted with a fluorescent sample. In these cases, substrate noise (though it exists) is irrelevant to the use of our products, because it does not contribute in any measurable way to the background reading of the reacted chip. In rare cases involving extremely low background samples or gene expression measurements of rare transcripts, substrate noise may approach sample noise in magnitude. In these cases, it may be desirable to use a substrate that has a lower intrinsic fluorescence and reflection, such as SuperAmine instead of SuperAldehyde.
For best results, please test our products in the context of REAL EXPERIMENTS rather than simply taking note of the fact that our Substrates manifest substrate noise that is greater than plain glass. ALL SUBSTRATES that contain an organic treatment or coating will produce some intrinsic fluorescence and reflection. Please also test our product “right out of the box” rather than waiting hours or days to measure the substrate noise. Fluorescent contaminants present in non-cleanroom air including cleaning agents, solvents used in marking pens, and hydrocarbon emissions from vacuum pumps, arrayers, centrifuges and other instruments can elevate the substrate noise reading considerably. Please also note that airborne particles including dust and other particulates greatly elevate the background reading because these particles are highly reflective in the presence of laser and white excitation light. Understanding the technical details of our products is important and we recommend that you commit these concepts to working memory as you proceed with your experiments.
Table. Substrate noise
|
Product |
Average substrate noise (ScanArray 3000) |
Maximum allowable substrate noise (ScanArray 3000) |
Average substrate noise (ScanArray Express) |
Maximum allowable substrate noise (ScanArray Express) |
|
SuperClean |
88 |
200 |
501 |
1,250 |
|
MirrorClean |
63 |
150 |
377 |
950 |
|
SuperEpoxy |
157 |
250 |
456 |
1,500 |
|
MirrorEpoxy |
114 |
475 |
348 |
1,125 |
|
SuperAmine |
229 |
500 |
861 |
2,000 |
|
MirrorAmine |
168 |
375 |
636 |
1,500 |
|
SuperAldehyde |
278 |
500 |
729 |
2,000 |
|
MirrorAldehyde |
217 |
375 |
568 |
1,500 |
 |
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 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).
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Technical Assistance
Please contact us if you have any comments, suggestions, or if you need technical assistance. By electronic mail: arrayit@arrayit.com (under the subject heading, please type, “Technical assistance”). By telephone: (408) 744-1331, Monday–Friday, PST 9:00am - 4:30pm. Please remember that we want to hear about your successes!
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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 Personal Microarrayer or NanoPrint 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.
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. Protein microarray data from the fluorescent image can be quantified, mined and modeled using many different commercial software packages. Including those made by BioDiscovery (Marina del Ray, CA), and many others make excellent products.
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 arrayit@arrayit.com for more information.
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 problems 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
Scientific Publications
Click here and here for recent scientific publications using ArrayIt® brand SuperEpoxy Microarray Substrates from TeleChem International, Inc.
Recommended Equipment and Reagents
NanoPrint™ Microarrayers
SpotBot® 2 Personal Microarrayers
InnoScan® Microarray Scanners
SpotLight™ Microarray Scanners
Microarray Hybridization Cassettes
High Throughput Wash Station
Microarray High-Speed Centrifuge
Protein Printing Buffer
BlockIt™ Blocking Buffer
Microarray Air Jet
Microarray Cleanroom Wipes
PCR Purification Kits
BlockIt Blocking Buffer
Micro-Total RNA Extraction Kit
MiniAmp mRNA Amplification Kit
Indirect Amino Allyl Fluorescent Labeling Kit
Universal Reference mRNA
Green540 and Red640 Reactive Fluorescent Dyes
Hybridization Buffers
Ordering Information
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