Microarray Solutions newsletter #4

The Nexterion® Newsletter © SCHOTT

The Nexterion® Newsletter

Microarray SolutionsEdition # 4 / September 2008

Editorial.........................................................2

New Aminosilane Slide.................................3Nexterion® Slide AStar

Protein Microarrays....................................4-5Selecting the optimal surface for your protein microarray

Antibody Arrays.............................................6Epitome Biosystems, Boston, MA: Ti-Tyr™ Profiling chips for detection and quantification of phosphorylation sites

News.............................................................7Latest news from SCHOTT

Meet us.........................................................8Conference and exhibition calendar

Competition...................................................8Win an iPod

2The Nexterion® Newsletter #4 September 2008 page 2

Editorial

Dear Reader,

Welcome to the latest edition of the SCHOTT “Microarray Solutions” newsletter. Since the last edition, there have been a number of developments at SCHOTT that we hope will continue to allow us to offer the latest microarray substrates and accessories. Some of these developments will be covered in this newsletter, including the launch of a new coated slide product, and SCHOTT’s continued focus on developing the best range of slide surfaces for protein microarrays.

When SCHOTT first entered the microarray slide business back in 2002, aminosilane coated substrates were by far the most popular choice for printing PCR products. Even after all this time, aminosilane still remains an extremely popular slide surface for printing DNA microarrays. As a result of this continued popularity, SCHOTT will soon launch what we feel will be the best aminosilane coated substrate in the market, Nexterion® Slide AStar. There is a short interview with Dr Dietrich in this newsletter that gives some insights into the background and development of the product.

Last year, SCHOTT introduced a range of new Nitrocellulose slides (Nexterion® Slide NC). These were jointly developed by SCHOTT and Sartorius Stedim to address the needs of the rapidly growing protein microarray market. The high level of customer interest in Nexterion® Slide NC, and other SCHOTT surfaces, led to our decision to give a presentation on “How to select the most appropriate protein microarray surface” at PepTalk 2008 in San Diego, CA. The talk was well received both by the delegates and the press. In this issue we look back on Dr. Dietmar Knoll’s talk, and hopefully offer some invaluable advice on key factors to consider when selecting the most appropriate slide surface.

This edition of “Microarray Solutions” will also introduce you to our exciting new product catalogue, and the newly developed web based “Frequently Asked Questions”. These will both help to complement the first class technical and customer support SCHOTT continues to strive to offer all our customers.

I hope that you will enjoy reading this edition of our newsletter and, as always, please feel free to contact us if you have any ideas for articles you would like to see in future editions. These could include having your facility and research work with Nexterion® product featured, or a suggestion for an application area that you would like us to focus on. Finally don’t forget to enter our competition to win an iPod Classic. Viel Glück!

Regards,

Christian Jabschinsky

© SCHOTT

Christian JabschinskyGeneral Manager

Nexterion – Microarray Solutions


NEW Nexterion® Slide AStar Taking Aminosilane Slide Performance to the Next LevelIn this article we discuss the development of the new aminosilane slide from SCHOTT with the Director of R&D, Dr. Rüdiger Dietrich.

MS: Why did SCHOTT decide to develop yet another Aminosilane coated slide?

RD: That’s a good question! Aminosilane slides still remain the most popular choice for printing both PCR products, and longer oligonucleotides. This is despite the widespread availability of potentially superior surface chemistries. To better address the needs of the current market, and in collaboration with colleagues in marketing, we developed a specification for a new “high performance” aminosilane slide (see table on left).

MS: What were seen as important parameters for the new slide?

RD: We agreed targets for performance characteristics such as a spot size with typical spotting buffer and conditions, required shelf lives of unprinted and printed slides, low background and high coating uniformity. Developing a commercial product is always a balance between creating the best product technically, and practical considerations like; are the reagents that we want to use readily availability for at least two suppliers at a reasonable cost? From the production point of view, a major consideration was that the slides must be able to be produced in a robust and stable process. Fortunately the seven years experience SCHOTT has with slide coating technologies was a major advantage in this respect. Alongside optimising the production process we have also developed new quality control for checking things like coating homogeneity.

MS: SCHOTT already offers two aminosilane coated slides, isn’t adding a third going to confuse customers?

RD: Yes, potentially this could be a problem, and for this reason we have decided, in future, just to offer two type of aminosilane slides, Nexterion® Slide A+, and Slide AStar. The current Nexterion® Slide A will still be available on request, but we will not be actively promoting it. We have also been able to improve the performance and shelf life of Slide A+ as a result of what we learned during the development of Slide AStar.

MS: Did you have any surprises during the development of the new product?

RD: We first evaluated samples of all the commercially available aminosilanes. These are available with varying linker chain lengths and numbers and positions of amino groups. We also put prototype coated slides through full DNA hybridization experiments to find out the best performing surface under varying conditions. The way the aminosilane coating works is that the negatively DNA is attached to the surface by ionic interaction with positively charged amine groups. We had always thought that longer aminosilane molecules with multiple amino-groups produce higher signal-to-noise ratios, as their higher net positive charge will bind more DNA to the slide surface. However, we found that the situation is actually more complicated, and that while surfaces with a higher charge do bind more DNA, they are also much more difficult to block effectively, resulting in significantly higher non-specific binding. The higher levels of non-specific binding have a negative effect leading to poorer signal-to-noise ratios. For this reason the aminosilane used for Slide AStar is a very short-chained molecule.

MS: When will the new Nexterion® Slide AStar be available?

RD: We have already started sampling large customers, but the official product launch will be at the Discovery-2-Diagnostics conference in San Diego on October 20 2008, so watch this space!

© SCHOTT

Specification for AStar Coating

• Contact angle 50°• Target spot size 100 microns

• Low fluorescence background (allowing long term storage of printed slide)

• Distinguished coating uniformity, CV’s < 5%

• Robust and stable (highly reproducible) coating process

• Economic production costs

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Dr. Dietmar Knoll

Senior R&D Scientist SCHOTT Microarray Solutions

Jena, Germany.

Prior to this, Dietmar worked for Quantifoil Micro Tools, a small biotech company acquired by SCHOTT in 2003. He received his Ph.D. in Organic Chemistry from Leipzig University and has extensive expertise in organic surface coatings.

SCHOTT Guide to Selecting the Optimal Slide Surface for your Protein Microarray

In January, Dr. Dietmar Knoll gave a presentation at the PepTalk 2008 conference in San Diego, CA, outlining some of the factors researchers should consider when selecting suitable surfaces for functional protein microarrays. The talk was well received, and was subsequently referenced in an article in Genetic Engineering News (15th March 2008: “Protein Microarray Uses Abound”). In the following article, Dr. Knoll discusses some of the key points from the talk with “Microarray Solutions”.

MS: Why has SCHOTT dedicated so many resources to developing a range of products for what is still a relatively small target market, when compared to DNA microarrays?

DK: Proteins are a very important class of bio-molecules to study as they participate in every process within the living cell ,for example, as enzymes, receptors, structural proteins, carrier proteins and so on. As a result, functional protein microarrays have become the fastest growing part of the microarray slide market, and are suited to a wide range of applications, such as biomarker discovery, antibody and protein substrate profiling, and protein quantification in diagnostic assays. Microarrays provide a way of doing this in a rapid, highly parallel, and cost effective way. However, when compared to DNA microarrays, there are still many technical challenges to overcome.

MS: What are some of the technical obstacles to creating function protein microarrays?

DK: One of the major challenges is the generation of the content itself. To ensure accurate results, a stable protein is necessary, and finding or purifying suitable high quality proteins can be costly in both time and money. Proteins are much more complex than DNA, starting with the higher number of building blocks to form polypeptides, continuing with a secondary structure, and finally a more or less susceptible 3-D structure, which is essential for their function. Because of the complexity of a protein, providing a stable environment to maintain the structure is important for any kind of biological assay. Consequently, slide surfaces play a vital role providing a support structure to keep the protein molecules intact and functioning properly, and at a fixed location for interaction with another biological molecule.

MS: What are the most important slide surface characteristics?

DK: Many of the slide surface characteristics important for DNA microarrays also hold true for protein microarrays. These include planar surface, high binding capacity, low non-specific binding, high sensitivity, low auto-fluorescence of the glass and compatibility with the process protocols. However, the stabilizing environment of the slide surface is of much greater importance for protein microarrays, along with the need for high binding capacity, tolerance to a wide range of print buffers, and the opportunity to analyze the arrays using label free detection systems.

MS: What types of slide surface are currently available from SCHOTT for protein microarrays?

DK: The SCHOTT coated glass substrates can be categorized into either two-dimensional (2-D) or three-dimensional (3-D) surfaces.

2-D Substrates provide a planar surface consisting of a chemical monolayer, which actively immobilize protein molecules via epoxy (Nexterion® Slide E), or aldehyde groups (Nexterion® Slide AL). These surfaces are normally very robust and reliable, however 2-D substrates provide very little structural support for the tertiary or quaternary structures of proteins. Consequently, this

© SCHOTT

“Because proteins have slightly different requirements to maintain their 3-D structure, for example membrane compared to cytosolic proteins, and not all requirements can be optimally reflected by one “universal” slide surface, so the user has to choose the most suitable slide surface solution for their specific application.”

Dr. Ruediger Dietrich, Director of Research & Development at SCHOTT Microarray Solutions.


type of surface is usually most suitable for arraying more “robust” proteins like antibodies, or in assays that do not require the protein probes to be functionality active. Another point to consider is the high steric hindrance with 2-D substrates. This disadvantage can be circumvented by addition of appropriate linkers to keep the probe away from the surface. The binding capacity of 2-D substrates is generally significantly less than with 3-D.

Using 3-D Substrates overcomes both limitations mentioned above as their multi-dimensional structure help to retain protein functionality and offer a high binding capacity. Another advantage of these substrates is that the open 3-D structure can provide the target with better accessibility to the printed probes. 3-D slide surfaces can be further divided into thin film and thick film coatings, thick films are typically tens of microns thick, whereas thin film typically have a thickness of less than a few hundred nanometres. Thin film coatings such as hydrogel slides (Nexterion® Slide H), Streptavidin-hydrogel (Nexterion® Slide H-S) and polymer slides (Nexterion® Slide P), have higher binding capacity than 2-D slides, and provide a exceptionally low non-specific binding after blocking. Therefore, high signal-to-noise ratios, and sensitivity can be achieved with these substrates. Examples of thick 3-D coatings are nitrocellulose coated slides (Nexterion® Slide NC).

MS: During your talk you mentioned reverse phase assays, do these require specific substrates?

DK: Yes, for reverse phase using cell lysates, the thin film hydrogel and polymer surfaces are not really suitable. These probe types require high concentrations of detergent to avoid precipitation, conditions that would result in very large spot sizes and poor spot morphology on hydrogel and polymer surfaces. Therefore the thick 3-D substrates like nitrocellulose coated slides (Nexterion® Slide NC) are the substrates of choice for this application. Generally, nitrocellulose slides are a good starting point for most protein array applications as they produce a linear correlation between probe and target over a wide concentration range. Another advantage is that for many applications printed nitrocellulose slides exhibit a very long shelf life at ambient temperatures without significant loss of activity. The disadvantages discovered when evaluating nitrocellulose surfaces can include high background, and no possibility of having additional layers such as, optically active layers under the opaque nitrocellulose coating for enhancing signal or use in label free detection.

MS: Do you have any final recommendations?

DK: The range of possible assays, technologies and studies involving analysis of proteins is very diverse, and there is no one “universal” surface that will work for all. Indeed the vast number of variables that can impact the quality of data a user can generate from a protein microarray dictates that even the above guidelines may not always be totally reliable. SCHOTT typically recommending that users try a variety of different slide surfaces rather than focus their efforts entirely on one slide surface. For additional advice on selecting the most appropriate slide surface for your protein application, or to receive a copy of the PepTalk 2008 presentation given by Dr. Dietmar Knoll, please send an e-mail message.

© SCHOTT

P r o b e t y p e

S u r f a c e t y p e

C o a t i n g p r o p e r t i e s

R e c o m m e n d e dN e x t e r i o n

p r o d u c t ®


Epitome Biosystems Antibody Arrays

Epitome Biosystems, Waltham, MA: Ti-Tyr™ Profiling Chips for Detection and Quantification of Site-Specific Phosphorylation via Sandwich ImmunoassaysEpitome Biosystems is pioneering innovative protein measurement products to enable drug discovery, drug development and the practice of clinical medicine. The company's highly quantitative and specific protein measurement products provide critical information on biological mechanisms and reveal important biomarkers or readouts of cell function. Epitome’s initial focus is on novel phosphorylation measurement products, including mass spectrometry (MS)-based biomarker discovery, as well as profiling chips and multiplex assay kits that are based on Epitome’s proprietary EpiTag™ technology. The EpiTag™ technology platform enables quantitative, multiplexed and highly specific measurements of protein abundance and amounts of phosphorylation at specific sites using targeted antibody pairs in a peptide-based sandwich immunoassay format. Epitome’s Ti-Tyr™ Chip provides quantitative profiling of dozens of phosphorylation sites simultaneously, on up to sixteen samples per chip, using a standard microarray reader. These products enable:

Quantitative assessment of phosphorylation at individual sites on proteins

Analysis of on- and off-target drug responses across signalling networks

Measurement of low copy number proteins and low abundance phosphorylation events

Epitome’s innovative protein measurement products and services enable Pharmaceutical, Biotech and Academic researchers to obtain highly specific, quantitative information about the cell-signalling network, which is a key interface for monitoring drug action and understanding disease. Key application areas include biomarker discovery and development, target validation and elucidation of mechanisms of drug action.

The Ti-Tyr™ Profiling Chip is a tyrosine phosphorylation focused product, based on Epitome’s proprietary EpiTag™ technology. Tyrosine phosphorylation is measured quantitatively at a site-specific level, after proteolytic fragmentation of samples. The Ti-Tyr™ Chip allows for multiplexed analysis across 74 phosphotyrosine sites on 60 different proteins using a planar microarray. The Ti-Tyr™ Chip covers a wide range of content including receptors (e.g. EGFR and HER2), adapter and scaffold proteins (e.g. IRS1 and Crk), intracellular mediators (e.g. Erk1 and Raf-1) and transcription factors (e.g. c-Jun and STAT3). A single chip can be used to analyze 16 different samples, including reference standards and controls, or four chips can be mounted in a carrier and treated similar to a microtiter plate.

A number of coated microarray slides from several manufacturers were evaluated on their ability to meet the demands of the Epitome application. “SCHOTT slides were the only slides that provided uniform, high signals and very low backgrounds necessary for our highly multipexed antibody arrays,” reported Tim Nadler, Director of Product Development, Epitome BioSystems

SCHOTT slide quality enabled Ti-Tyr™ Chip’s ability to consistently perform quantitative, high content measurements.

EpiTag™ technology provides a unique, powerful platform for targeted, quantitative and highly multiplexed protein measurements. The approach provides a predictable, streamlined path for assay development and enables rapid content generation. In addition to ongoing Ti-Tyr™ Chip content expansion, similar profiling chips incorporating threonine and serine phosphorylation measurements are in development.

© SCHOTT

Company: Epitome BiosystemsLocation: Waltham, MAWeb: www.epitomebiosystems.comSubstrate Used: Nexterion® Slide H


SCHOTT Microarray Solutions News

New SCHOTT Microarray FAQ Section Goes Online

Since entering the microarray substrate market several years ago, SCHOTT has aimed to provide customers with the best possible technical support, whether it be through direct contact with designated technical support specialists, or via support materials such as protocols, scientific papers or customer references. To further enhance this offering, SCHOTT has just launched a new Frequently Asked Questions (FAQ) section to be found at the Nexterion® web site.

New Nexterion® Microarray Substrates Catalogue

The new catalogue for the Nexterion® range of microarray products will be available from 01 September 2008. The format of the new catalogue will be a flexible ring binder system, allowing customers to keep all the details of the Nexterion® products, such as product flyers, ordering details, protocols etc., in one place. The catalogue can easily be kept up to date simply by adding new information sheets as they become available. The layout of the individual product flyers has been redesigned to improve readability and allow customers to find the important information quickly.

Nitrocellulose Slide Application Note

SCIENION AG manufactures and markets non-contact dispensing systems for printing microarrays. They have recently published an application note favourably comparing the new SCHOTT Nexterion® nitrocellulose slides (Slide NC-W and NC-D) to the market leading nitrocellulose slide. For further details please download the “sciFLEXARRAYER Application Note No. 08001” from:

www.scienion.de/innolino/pdfdownload.php?id=80

Nexterion® MPX 16-well Slide Price Reduction

The Nexterion® MPX 16-well slides have always been a popular way to run multiple arrays on one slide. SCHOTT has now made these slides even more cost effective by reduced the list price of all the MPX slides. In order to do this the slide package now contains five MPX slides rather than eight, and the Nexterion® 16-well self-adhesive superstructure and sealing strip are now supplied as a separate item.

For more details about the revised MPX slide range please visit the SCHOTT Nexterion® web site.

© SCHOTT


Conference and Exhibition Calendar 2008/09

Exhibition Location DateDiscovery2Diagnostics Manchester Grand Hyatt

San DiegoOne Market PlaceSan Diego, CA 92101 USA

October 20-22 2008

PEPTALK 2009 Hotel Del Coronado1500 Orange AveCoronado, CA 92118 USA

January 12-16 2009

LabAutomation2009 Palm Springs Convention Center, Palm Springs, CAUSA

January 25-27 2009

Statusseminar Chip Technologies

DECHEMA-HausTheodor-Heuss-Allee 2560486 Frankfurt am Main, Germany

March 05-06 2009

Advances in Microarray Technology

Stockholm International FairsS-125 80 STOCKHOLMMässvägen 1, Stockholm/Älvsjö, Sweden

May 19-20 2009

Did you know?

By entering our competition, you will have a chance to win one Apple iPod classic (80 GB).Simply answer the three questions below, and send your entry to arrive before 12 midnight (CET) on 28 February 2009. By email: [email protected] fax: +1-502/966-4976The winner will be the first correct entry drawn at random.

Name : _____________________________________________

Email : _____________________________________________

Institute/Company : _____________________________________________

1. What title was the city of Jena awarded in 2008?□ City of Optics 2008 - "Stadt der Optik"□ City of Science 2008 - "Stadt der Wissenschaft"□ City of Sport 2008 - "Stadt des Sports"

2. Which Nexterion® slide does Epitome Biosystems use for their profiling chips?□ Nexterion® Slide E□ Nexterion® Slide H□ Nexterion® Glass B

3. What is the name of the new aminosilane slide from SCHOTT?□ ASpar□ APlug□ AStar

* Only one entry per person. The judges' decision is final. This competition is void where prohibited. This competition is run and staged in accordance with German legislation.

© SCHOTT

Microarray Solutions

SCHOTT North America Inc.

5530 Shepherdsville Road

Louisville, KY 40228

USAPhone: +1- 502-657-4417Fax: +1- 502-966-4976E-Mail: [email protected]/nexterion

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