Instruction Manual for Protein Microarrays - Greiner Bio … Manuals/0506... · I. Introduction...

Instruction Manual for Protein Microarrays

Last update: 2005 - 06

I. Introduction

Greiner Bio-One and SCIENION have developed an integrated and complete system for the

production and use of protein microarrays. Their combined expertise in injection moulding,

surface modifications, chemistry and molecular biology results in a unique polymer-based

microarray platform with outstanding quality and performance.

Greiner Bio-One’s HTA™Slides are manufactured under very stringent

conditions and controls that guarantee their high quality (clean, flat, planar).

In collaboration with PolyAn a proprietary technology is used to produce

novel, three dimensional surfaces with excellent specific binding properties.

SCIENION Buffer Systems offer optimized solutions for protein microarray production and

use. sciSPOT-PROTEIN is a unique spotting solution which facilitates a uniform binding of

proteins, antibodies and peptides to HTATM Slides. sciBLOCK buffer removes unbound

proteins after spotting and blocks the HTATM surfaces during incubation. sciBIND buffer

creates the most favourable environment for binding reactions. Finally, sciWASH-Pro buffer

is a microarray wash buffer which is formulated to promote specific binding and to reduce

background signals.

II. Product description

A. Greiner Bio-One HTA™Slides

Product Slides per case Cat.-No.

HTA™Slide1, Multifunctional 25 445 825

HTA™Slide1, 3-D Aldehyde 5 445 840


The HTA™Slide1 has the dimensions of a standard microscope slide (25 x 75 x 1 mm).

Barcode-labelling is offered upon request.

Product Slides per case Cat.-No.

HTA™Slide12, Multifunctional 5 446 820


The HTA™Slide12 has been designed for high-throughput, e.g. for routine diagnostic

applications. The HTA™Slide12 is divided into 12 shallow compartments. Each compartment

has a printable area of 6 x 6 mm and a low well rim of only 0.5 mm. Printing devices (either

contact or non-contact) can move quickly without significant movement in the z-axis, allowing

‘on the fly’-printing. With the HTA™Slide12 it is feasible to process 12 different samples

simultaneously. The slides increase the speed of analysis and lower the costs significantly

without risking cross-contaminations. The slides have the standard format of 25 x 75 x 1 mm

and can be scanned with all commercially available scanners. Barcode-labelling is offered

upon request.

Storage Conditions: Packaged HTA™ Slides should be stored in the dark at room

temperature (20-27°C) and used prior to expiration date. Once the package has been

opened, slides should be used within 1 week if stored at room temperature under inert

condition inside a desiccator and protected from light.

Disclaimer: Greiner makes no representation or warranty, express or implied,

irrespective of the legal grounds, that the products supplied under this contract do not

infringe intellectual property rights of third parties. Greiner is not obligated or

responsible to defend or to indemnify anyone against any claim or lawsuit for the

infringement of any such intellectual property right of third parties. The contractual

partner acknowledges that the avoidance of such infringement of intellectual property

rights of third parties will remain the responsibility of the contractual partner.

B. SCIENION Buffer Systems

Product Volume (ml) Cat.-No.

sciSPOT-PROTEIN, 2x conc. 25 445 055

The spotting buffer sciSPOT-PROTEIN is an advanced buffer system containing a mixture of

ionic and polymeric materials. sciSPOT-PROTEIN buffer has been optimized for spotting

proteins, antibodies and peptides to Multifunctional and 3-D Aldehyde HTA™Slides. Use of

sciSPOT-PROTEIN will improve the quality of microarrays prepared by contact and non-

contact protein printing technologies.

Users will appreciate the following features of sciSPOT-PROTEIN:

Supports multiple printing technologies (contact and non-contact printing)

Prints proteins, antibodies and peptides

Stabilizes protein samples and prevents denaturation

Provides uniform coupling and feature size

Improves data analysis by excellent spot geometry

Arrives pre-mixed as a 2x solution, sterile and protease-free


sciBLOCK, 5x conc. 500 445 052

The sciBLOCK buffer has been designed to remove unbound protein molecules after

spotting and to block the HTATM surface simultaneously.

Users will appreciate the following features:

Ultra-pure reagents used for buffer formulation

All buffers purified by sterile filtration

Provided pre-mixed as a 5x concentrate

Can be used for protein, antibody and peptide microarray application



sciBIND, 2x conc. 1.6 445 054

sciBIND is an advanced protein incubation buffer containing salts and pH stabilizers.



Increases the signal by accelerating binding kinetics

Increases the sensitivity by reducing background fluorescence

Provides a uniform binding layer

Buffering components stabilize extended reactions



sciWASH-Pro, 8x conc. 500 445 051

sciWASH-Pro is an advanced washing buffer containing salts, pH stabilizers, and polymers

to remove unspecifically bound proteins from protein microarrays.



Increases the signal by accelerating the binding kinetics

Arrives pre-mixed, sterile and protease-free, no preparation required

Storage Conditions: All components of the SCIENION Protein Buffer Set should be stored

at 4°C. For long periods store the buffer at -20°C. All components of the SCIENION Protein

Buffer System have a 1-year shelf life when handled and stored properly.

Safety Considerations: When working with SCIENION Buffer Systems please follow all

generally accepted laboratory safety guidelines. At a minimum, wear appropriate personal

protective equipment such as a lab coat, safety glasses, powder-free latex gloves, etc.

Follow recommended standard operating procedures for any laboratory equipment used in

your experiments.

Product Use Limitations, Warranty, Disclaimer: SCIENION Buffer Systems have been

scientifically developed and are sold for research purposes only. SCIENION Buffer

Systems are not for use in human diagnostics or for drug purposes. Extreme care and close

attention should be practiced in the use of the materials described herein. Please refer also

to the material safety data sheets. All SCIENION products are subject to extensive quality

control and are guaranteed to perform as described when used properly. Any problems with

any SCIENION product should be reported to SCIENION immediately. SCIENION’s liability is

limited to the replacement of the product, or a full refund. Any misuse of this product is the

full responsibility of the user, and SCIENION makes no warranty or guarantee under such

circumstances.

III. Materials provided by the researcher

Slide staining racks and glass staining jar (recommended: Thermo Shandon, Cat.-

No.121)

Shaking incubator

Distilled water

Centrifuge with microplate carriers (to spin-dry slides)

Microcentrifuge

Glass cover slips

Hybridization chamber (recommended: sciHYBCHAMBER)

Watchmaker forceps

Clean room wipes

IV. Protein Array Preparation and Binding Reactions

A. Printing

1. Obtain 0.2-1.0 µg/µl of protein samples.

Protein samples should be free of aggregates and precipitates to prevent clogging of printing

devices or impairing attachment to the microarray substrate. Users may want to test a range

of different concentrations to determine the optimal target concentration for a particular

assay, though the values given above work very well for many different applications.

2. Transfer 4 µl of each protein sample into a 96- or 384-well microplate.

Certain proteins are fragile and protein samples should be handled with care to avoid

damaging the protein structure and function.

3. Add 4.0 µl sciSPOT-PROTEIN to each 4.0 µl protein sample.

This will give a final concentration of 1x sciSPOT-PROTEIN and a final protein spotting

concentration of 0.1-0.5 µg/µl. Most proteins or protein extracts are more stable at 4°C.

Keeping the protein samples cool will improve stability.

4. Mix the samples by pipetting up and down 10 times.

This step ensures that the protein samples mix thoroughly with the 2x sciSPOT-PROTEIN

buffer. Failure to mix the samples thoroughly will produce poor quality microarrays.

5. Print protein samples onto HTA™Slides by placing the carriers on a suitable microarraying device.

The optimal printing environment is a temperature of 20°C and a relative humidity of 45-60%.

After printing allow the substrate to dry at room temperature for 15-30 min. The drying step

can be carried on the plate of the microarrayer or in slide boxes with the lid slightly open.

B. Blocking and Processing

Note: Do not allow the surface to completely dry at any time until you are ready to scan the

slide.

Prior to use dilute the sciBLOCK 5x buffer and the sciWASH-Pro 8x buffer with distilled

water. We recommend blocking and washing 5 slides in a glass staining jar (Thermo

Shandon Cat.-No.121), which requires 400 ml of buffer for each step in the protocol. If you

use alternative wash vessels, please adjust your volumes accordingly. Due to their low

density HTA™Slides will float in aqueous solutions. Therefore, place a piece of metal or

something equivalent on top of the slides.

1. Transfer printed HTA™Slides to a wash station containing sciBLOCK and incubate the slides at room temperature for 30-60 min with vigorous agitation.

Once the printing process is complete, wash the printed microarrays to remove unbound

protein molecules and components of the protein printing buffer. Protein binding to the

surface is extremely stable and the microarrays can be washed, blocked and reacted without

loss of coupled protein.

2. Transfer HTA™Slides to a wash station containing sciWASH-Pro and wash three times at 20-25°C for 10 min with agitation.

After blocking, wash the microarrays to remove excess sciBLOCK buffer. Make sure

sciWASH-Pro is moving smoothly across the microarray surface.

After washing the microarray with sciWASH-Pro, allow the liquid to pass off from the slide by

gently flicking, but do not allow the surface to completely dry. The processed microarrays

containing coupled target proteins can be reacted with fluorescent samples to study protein-

protein interactions.

C. Binding reaction

Note: Do not allow the surface to completely dry at any time after blocking the slide.

1. Transfer 10 µl of each protein sample into a reaction tube.

Sample transfer can be performed manually, with a multi-channel pipetting device, or with a liquid-handling system.

2. Add 10 µl sciBIND to each 10 µl protein sample.

Make sure that the transfer volume of sciBIND is 10 µl for all samples.

Note: Typically, the optimal concentration of the labelled detection reagent should be

between 0.01-1.0 µg/ml. However, some optimisation tests should be performed to

determine the best possible concentration of the labelled detection reagent before running an

assay. Additionally, the incubation period should be optimised for each system; typical

incubation times range from 30 min to 3 hrs.

3. Mix the samples thoroughly by pipetting up and down 10 times prior to application on the array.

4. Transfer the HTA™Slide to a sciHYBCHAMBER containing 10 µl of 2x SSC in each well.

5. Apply the protein sample to the microarray:

For HTA™Slide1: use 1 µl of protein sample in sciBIND per cm2 glass cover slip.

For HTA™Slide12: use 20 µl of protein sample in sciBIND per well.

Covering the wells of an HTA™Slide12 is not necessary. In order to avoid well-to-well cross-

contamination do not use a cover slip and handle HTA™Slides12 and the hybridization

chamber carefully once the probe has been applied.

6. Close sciHYBCHAMBER by tightening the four screws (cross-over) and incubate for 60 min at room temperature.

7. Transfer HTA™Slides to a wash station containing sciWASH-Pro and wash the HTA™Slides for 10 min at room temperature to remove unreacted detection material.

Following incubation with the detection reagent, allow the excess liquid to run off the slide by

gently flicking or carefully pipetting, and then incubate the slides in sciWASH-Pro wash buffer

with gentle agitation.

8. Transfer HTA™Slides to a wash station containing fresh sciWASH-Pro and wash at 20-25°C for 10 min with gentle agitation.

9. Repeat step 8 one more time.

10. Immediately dry the slide using an air or nitrogen stream to remove salt residue that is potentially visible on the array image.

If a cloudy or spotty background remains on the dried slide, briefly rinse again with deionised

water to remove dried salts. Immediately repeat the drying step by applying an air or nitrogen

stream.

11. Scan the microarray to produce the microarray image.

The dried slide can be read with any standard fluorescence-based microarray scanner.

Technical assistance

Please contact us if you have any comments or suggestions, or if you need technical

assistance:

Greiner Bio-One GmbH Maybachstr. 2

D-72636 Frickenhausen

Germany

By electronic mail: [email protected]

By telephone: (+49) 7022 948-0

Instruction Manual for Protein Microarrays - Greiner Bio … Manuals/0506... · I. Introduction...

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Transcript of Instruction Manual for Protein Microarrays - Greiner Bio … Manuals/0506... · I. Introduction...