Peptide Microarray Printing Buffers, Two Buffers (50 ml of 2X)

Arrayit Peptide Microarray Printing Buffers, Two Buffers (50 ml of 2X) is an advanced two-buffer system containing a proprietary mixture of organic solvents and ionic and polymeric materials for high quality peptide microarray printing. This advanced two-buffer formulation enhances peptide microarray manufacturing on Arrayit SuperEpoxy 2 and SuperEpoxy 3 Microarray Substrate Slides and other surface chemistries used for peptide microarrays. Peptide microarrays have numerous applications in research, antibody and microarray testing, life sciences, immunology and other areas. Arrayit PEP is supplied as a two-buffer system, 50 ml of 2X concentration for both buffers.

Buffers and Solutions - Peptide Microarray Printing Buffers to Enhance Peptide Microarray Manufacturing for Microarray Testing, Life Sciences, Research, Immunology and Other Applications

Arrayit Peptide Microarray Printing Buffers, Two Buffers (50 ml of 2X) is an advanced peptide microarray buffer system containing solvents, viscosity enhancers, stabilizers, and buffering components to increase the quality and stability of peptide microarrays by improving sample surface properties during printing. Arrayit Peptide Microarray Printing Buffers (Cat. PEP) are formulated to provide high coupling efficiency and unparalleled spot morphology with synthesized and modified peptides printed on Arrayit microarray slides and other surfaces that couple peptides in a covalent manner. This two-buffer kit is supplied as two 50 ml volumes of 2X solution.

Table of Contents

• Introduction
• Quality Control
• Product Description
• Technical Note
• Short Protocol
• Complete Protocol
• Recommended Equipment
• Troubleshooting Tips
• Technical Support
• Scientific Publications
• Ordering Information
• 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 Peptide Microarray Printing Buffers.

Quality Control
Arrayit assures the performance of this product. The finest scientific research was used to develop this product. Rigorous quality control monitoring on a lot-by-lot basis guarantees that the ingredients conform to the highest industry standards.

Product Description
Arrayit Peptide Microarray Printing Buffers (Cat. PEP) are an advanced buffering system containing a proprietary mixture of solvents, buffering components and stabilizers to enhance peptide microarray re-suspension post synthesis and printing. Arrayit PEP will increase the quality of peptide microarrays prepared using contact printing technology.

Arrayit Peptide Microarray Printing Buffers (Cat. PEP) offer the following user advantages:

• Supports multiple printing technologies including contact printing and ink-jetting
• Print unmodified and modified peptides
• Washes away during blocking and processing
• Manufacture peptide microarrays for a wide variety of applications
• Stabilizes peptide samples and prevents dehydration
• Provides uniform microarray feature size and coupling density
• Promotes peptide coupling to the microarray substrate slides
• Protects printed samples from environmental damage
• Slows sample evaporation within the source microplates
• Minimizes sample drying and crystallization on substrate slides and pins
• Washes away easily, leaving pure bound peptide molecules
• Improves deposition uniformity which facilitates data analysis
• Arrives pre-mixed as two 50 ml volumes of 2X solutions, sterile and protease-free
• Sufficient to print 50 million protein features (5,000 10K peptide microarrays)

Technical Note
Peptides can be modified with aminooxyacetic acid (AOA) at the amino terminus to allow covalent attachment of peptides to Arrayit SuperAldehyde 2, SuperAldehyde 3, SuperEpoxy 2 and SuperEpoxy 3 Microarray Substrate Slides (Cat. SMA2, SMA3, SME2, and SME3). The AOA primary amine couples to reactive aldehyde and epoxide groups using Schiff's base chemistry.

Figure 1. Arrayit peptide microarrays manufactured using Peptide Microarray Printing Buffers (Cat. PEP) can be reacted with fluorescent antibodies and other binding reagents in a two-color format and scanned for fluorescence emission at 550 and 650 nm.

Figure 2. Arrayit peptide microarrays manufactured using Peptide Microarray Printing Buffers (Cat. PEP) were printed on Arrayit SuperEpoxy 3 Microarray Substrate Slides (Cat. SME3) and scanned for fluorescence emission.

Figure 3. Arrayit peptide microarrays manufacturing using Peptide Microarray Printing Buffers (Cat. PEP) demonstrate high binding specificity with fluorescent antibodies. Peptides containing FLAG peptide epitopes produce strong signals and low background with an anti-FLAG antibody compared to an antibody specific for cellular protein Cluster of Determination 28 (CD28).

Figure 4. Arrayit peptide microarrays manufacturing using Peptide Microarray Printing Buffers (Cat. PEP) to re-suspend an overlapping peptide library can be reacted with a primary antibody, followed by a biotinylated secondary antibody and a streptavidin staining reagent containing fluorescent tags. A microarray scanner set to the green (532 nm) channel can be used for detection.

Figure 5. Arrayit peptide microarrays manufacturing using Peptide Microarray Printing Buffers (Cat. PEP) to re-suspend an overlapping peptide library can be reacted with human serum to allow binding of serum antibodies, followed by staining with a secondary antibody of the appropriate class (IgG, IgM, IgA or IgE) bearing fluorescent tags.

Short Protocol (Steps 1-10)
1. Synthesize AOA-modified peptides.
2. Re-suspend peptides at 1 mg/ml in 2X Peptide Printing Buffer PEP1.
3. Add an equal volume of 2X Peptide Printing Buffer PEP2.
4. Mix the peptides by pipetting up and down 10 times.
5. Print peptides onto SuperAldehyde 2, SuperAldehyde 3, SuperEpoxy 2, or SuperEpoxy 3 Microarray Substrate Slides.
6. Block the printed peptide microarrays using ChemBlock Blocking Buffer.
7. React the peptide microarrays with whole blood samples and stain.
8. Wash the peptide microarrays to remove unreacted fluorescent material.
9. Scan the peptide microarrays to produce a fluorescent image.
10. Quantitate and model the peptide microarray data.

Complete Protocol (Steps 1-10)
1. Synthesize AOA-modified peptides. Synthesize synthetic peptides for the peptide microarrays using the smallest synthesis scale available. Make sure to add an aminooxyacetic acid (AOA) modification to the amino terminus to allow covalent coupling to the slide surface.

2. Re-suspend peptides at 1 mg/ml in 2X Peptide Printing Buffer PEP1. The dry peptides in the bottom of each tube or microplate well should be re-suspended at 1 mg/ml in 2X PEP1 Peptide Microarray Printing Buffer. This can be performed by manual by pipetting or with a liquid handling robot. Care should be taken to avoid well-to-well contamination of the peptides.

3. Add an equal volume of 2X Peptide Printing Buffer PEP2. Adding an equal volume of PEP2 buffer will dilute the peptides to from 1.0 mg/ml to 0.5 mg/ml,, which is a good starting concentration for many applications. Optimization can be performed to identify the exact peptide concentration that is optimal for each assay.

4. Mix the peptides by pipetting up and down 10 times. This step ensures that the peptide samples mix thoroughly with the 2X Peptide Microarray Printing Buffers PEP1 and PEP2. Failure to mix the peptide samples thoroughly prior to printing will reduce the quality of the printed microarrays.

5. Print peptides onto SuperAldehyde 2, SuperAldehyde 3, SuperEpoxy 2, or SuperEpoxy 3 Microarray Substrate Slides. Peptides have been found to covalently coupled efficiently to Cat. SMA2, SMA3, SME2 and SME3 slides through Schiff's bases that form between the peptide AOA groups and aldehyde or epoxy groups on the microarray surface. Following printing, the microarray spots should be dried completely to drive the coupling reaction. Elevated temperatures and vacuum conditions can accelerate peptide microarray drying.

6. Block the printed peptide microarrays using ChemBlock Blocking Buffer. Just prior to use, the peptide microarrays should be blocked using Arrayit ChemBlock Blocking Buffer (Cat. CHE). Three 5 minute blocking cycles using ChemBlock Blocking Buffer in Arrayit Microarray Reaction Trays (Cat. MRT) is usually sufficient to remove unbound peptides and block the microarray surface for the binding reaction. The peptide microarrays should be washed three times for 1 min per wash with Protein Microarray Wash Buffer (Cat. PMWB) to remove the blocking solution.

7. React the peptide microarrays with whole blood samples and stain. Suspend a dried blood spot or whole blood sample in Dry Blood Reaction Buffer (Cat. DBSR) and mix well. Pipette the whole blood sample onto the peptide microarray slide and react for 30 min at 37°C to allow binding reactions between the bound peptides and antibodies in solution. Binding reactions can be performed using Arrayit Microarray Reaction Trays (Cat. MRT). After the binding step, wash the peptide microarray slide 5 times for 1 min per wash at room temperature to remove unbound peptides. After the wash step, stain the peptide microarray containing bound antibody using a suitable flourescent reagent such as a secondary fluorescent antibody diluted 1:1,000-1:10,000.

8. Wash the peptide microarrays to remove unreacted fluorescent material. Once binding between the bound peptides and the fluorescent staining reagent is complete, wash the peptide microarray slides to remove the unbound staining reagent. Washes can be performed three times for 5 min each at room temperature in Protein Microarray Wash Buffer (Cat. PMWB). After the washes, excess wash buffer should be removed from the surface by tapping or by centrifugation with an Arrayit Microarray High-Speed Centrifuge (Cat. MHC).

9. Scan the microarray to produce a fluorescent image. The fluorescent peptide microarrays can be scanned a high quality commercial scanning instrument such as the Arrayit InnoScan 710AL or GenePix microarray scanners from Molecular Devices. Scanner settings including laser, PMT, detection channel and resolution should be adjusted to optimize the scanned images.

10. Quantitate and model the peptide microarray data. Peptide microarray data from the fluorescent scans can be quantified, mined and modeled using many different commercial software packages including MAPIX® from Arrayit InnoScan and GenePix Pro® from Molecular Devices.

Recommended Products
Hybridization Cassettes
High-Throughput Wash Station
Microarray High-Speed Microarray Centrifuge
Arrayit Blocking Buffers
Nanoprint™ 2 Microarrayers
946 Microarray Printing Technology
SpotBot® 4 Personal Microarrayers
InnoScan® Microarray Laser Scanners
SpotLight™ 2 Microarray Scanners
SpotWare™ Colorimetric Microarray Scanners

Troubleshooting Tips
Reduced printing quality:

• Incomplete mixing of peptides in Peptide Microarray Printing Buffers PEP1 and PEP2
• Sub-optimal printing environment (55% humidity and 20°C recommended
• Sub-optimal microarray printing pin washing

Reduced protein coupling:

• Sub-optimal surface chemistry (SuperAldehyde and SuperEpoxy series slides recommended)

Weak fluorescent signals:

• Fluorescent staining reagent sub-optimal
• Washes too harsh