Arrayit Protein Printing Buffer (PPB) is an advanced buffering system containing a proprietary mixture of ionic and polymeric materials. Use of our Protein Printing Buffer will increase the quality of protein microarrays prepared using any contact or non-contact printing technology. Users will appreciate the following features of Protein Printing Buffer (PPB): supports multiple printing technologies (contact printing and ink-jetting), print proteins, enzymes, antibodies, receptors, antigens, and peptides, washes away during blocking and processing, manufacture microarrays of pure proteins, recombinant proteins and cellular extracts, stabilizes protein samples and prevents denaturation, stabilizes protein samples and prevents denaturation, provides uniform feature size and coupling density, promotes protein coupling to the microarray substrate, protects printed samples from environmental damage, slows sample evaporation within the source microplates, minimizes sample drying and crystallization on substrates and pins, washes away easily, leaving pure bound protein molecules, improves deposition uniformity which facilitates data analysis, arrives pre-mixed as 50 ml of 2X solution, sterile and protease-free, sufficient to print 50 million protein features (5,000 10K protein arrays).
Reagents - Microarray Buffers - Protein Microarray Printing Buffer for Protein Microarray Manufacturing
Arrayit offers advanced microarray print buffers containing mixtures of solvents, viscosity enhancers, stabilizers, and buffering components to increase the quality and stability of protein and peptide microarrays by improving sample surface properties during printing. Protein and Peptide Printing Buffers are formulated to provide high coupling efficiency and unparalleled spot morphology with proteins, antibodies, peptides, and other polypeptides by providing sample conditions that mimic the intracellular environment. Compatible with SuperEpoxy 2, SuperEpoxy 3 and many other surface chemistries. Supplied as 50 ml volumes of 2X solution.
Table of Contents
- Introduction
- Quality Control
- Product Description
- Technical Note
- Technical Assistance
- Short Protocol
- Complete Protocol
- Recommended Equipment
- Troubleshooting Tips
- 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 Protein Printing Buffer.
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 ingredients conform to the highest industry standards.
Product Description
The Arrayit Protein Printing Buffer (PPB) is an advanced buffering system containing a proprietary mixture of ionic and polymeric materials. Use of our Protein Printing Buffer will increase the quality of protein microarrays prepared using any contact or non-contact printing technology.
Users will appreciate the following features of Protein Printing Buffer (PPB):
- Supports multiple printing technologies (contact printing and ink-jetting)
- Print proteins, enzymes, antibodies, receptors, antigens, and peptides
- Washes away during blocking and processing
- Manufacture microarrays of pure proteins, recombinant proteins and cellular extracts
- Stabilizes protein samples and prevents denaturation
- Provides uniform feature size and coupling density
- Promotes protein coupling to the microarray substrate
- Protects printed samples from environmental damage
- Slows sample evaporation within the source microplates
- Minimizes sample drying and crystallization on substrates and pins
- Washes away easily, leaving pure bound protein molecules
- Improves deposition uniformity which facilitates data analysis
- Arrives pre-mixed as a 2X solution, sterile and protease-free
- Sufficient to print 50 million protein features (5,000 10K protein arrays)
Technical Note
It may be necessary to add protease or phosphatase inhibitors to the Protein Printing Buffer to increase the stability of certain proteins or protein extracts. Protein stability can also be enhanced if microarray manufacture is performed at 4°C. The SpotBot Personal Microarrayer can be moved into a cold room easily, and operated at 4°C. A special “cold platen” is also available for the SpotBot® 2 if a 4°C printing temperature is required in an ambient laboratory environment. Please contact arrayit@arrayit.com for technical details.
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 email: arrayit@arrayit.com, Monday–Friday, PST 8:00am - 8:00pm. Please remember that we want to hear about your successes!
Figure 1. 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 or NanoPrint running SMP9 Micro Spotting Pins. Printed microarrays were blocked for 30 min with 1X phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), then 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.
Short Protocol (Steps 1-10)
1. Obtain 0.2-1.0 µg/µl protein samples.
2. Transfer 4.0 µl per well of each protein sample into 96- or 384-well microplates.
3. Add 4.0 µl per well of 2X Arrayit Protein Printing Buffer (PPB).
4. Mix the samples by pipetting up and down 10 times.
5. Print protein samples onto SuperEpoxy, SuperAldehyde, SuperNitro or SuperAmine.
6. Use BlockIt to block and process the printed protein microarrays.
7. React the processed microarrays with fluorescent samples.
8. Wash the microarrays to remove unreacted fluorescent material.
9. Scan the microarray to produce a fluorescent image.
10. Quantitate and model the fluorescent data.
Complete Protocol (Steps 1-10)
1. Obtain 0.2-1.0 µg/µl protein samples. Phosphate buffered saline (PBS) at 1X concentration works very well for most proteins. 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 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.
2. Transfer 4.0 µl per well of each protein sample into 96- or 384-well microplates. Transfer can be performed manually by pipette or with a liquid handling robot. Certains proteins are fragile and protein samples should be handled with care to avoid damaging the structure and function of proteins.
3. Add 4.0 µl per well of 2X Arrayit Protein Printing Buffer (PPB). This will give a final concentration of 1X Protein Printing Buffer and a final protein concentration of 0.1-0.5 µg/µl. Certain proteins or protein extracts are more stable at 4°C. Keeping the proteins samples cool may improve stability. Stability can also be improved in some cases by the addition or protease and phosphatase inhibitors or by the use of a SpotBot 2 Personal Microarrayer equipped with a cooled platen.
4. Mix the samples by pipetting up and down 10 times. This step ensures that the protein samples mix thoroughly with the 2X Protein Printing Buffer. Failure to mix the samples thoroughly will produce poor quality microarrays.
5. Print protein samples onto SuperEpoxy, SuperAldehyde, SuperNitro or SuperAmine Substrates. The SuperEpoxy surface seems to couple proteins more readily than SuperAldehyde or SuperAmine, owing to the increased reactivity of the epoxide groups. Another advantage of SuperEpoxy is that coupling can occur in humid conditions as may be required to maintain protein structure and function. Other users have gotten excellent results with SuperAldehyde and SuperNitro. The best advice for protein printing is to start with SuperEpoxy and then explore the other surfaces for optimal performance once the SuperEpoxy experiments are working. Also compatible with surfaces from other vendors.
6. Use BlockIt to block and process the printed protein microarrays. Once the printing process is complete, wash the printed microarrays with BlockIt to remove unbound protein molecules and components of the Protein Printing Buffer. Protein binding to the SuperEpoxy surface is extremely stable and the microarrays can be washed, blocked and reacted without sufficient loss of coupled protein. Coupling to SuperEpoxy occurs within 10 minutes after printing, and blocking can be performed almost immediately after printing. A good blocking protocol involves a 30-minute incubation at room temperature in 1X BlockIt, using a High Throughput Wash Station or an equivalent device. Blocking should be performed with very gentle buffer agitation and the stir plate set on a low speed. The BlockIt blocking step will couple BlockIt proteins to unreacted epoxy groups and prevent background fluorescence. After blocking, wash the microarrays to remove the excess BlockIt. Washes can be accomplished three times for 2 min each at room temperature with 1X PBS, using a High Throughput Wash Station or an equivalent device. Washes should be performed with very gentle buffer agitation and the stir plate set on a low speed to avoid damaging the proteins.
7. React the processed microarrays with fluorescent samples. Processed microarrays containing coupled target proteins can be reacted with fluorescent samples to study protein-protein interactions. Binding reactions can be performed using BlockIt or buffers containing a buffered salt solution and blocking protein, and probes such as fluorescent antibodies diluted 1:1,000. Fluorescent samples can be incubated as a droplet over the printed microarray, underneath a cover slip, or in a microfluidics chamber. A 60-minute incubation at room temperature is usually sufficient to obtain strong binding and intense fluorescent signals (see Fig. 1). A Hybridization Cassette can be used to prevent sample evaporation during prolonged binding reactions.
8. Wash the microarrays to remove unreacted fluorescent material. Once binding between the bound target proteins and the fluorescent protein probe molecules is complete, wash the microarray to remove the unbound material. Washes can be performed three times for 5 min each at room temperature in 1X PBS. After the wash procedure, excess buffer should be removed from the surface by tapping or by centrifugation with a Microarray High-Speed Centrifuge.
9. Scan the microarray to produce a fluorescent image. The fluorescent microarray can be scanned or imaging using any of a number of high quality commercial detection instruments from Perkin Elmer (Boston, MA), Virtek (Ontario, Canada), Axon (Union City, CA), and many others. Instrument settings can be adjusted to optimize the imagine process.
10. 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. Perkin Elmer (Boston, MA), Virtek (Ontario, Canada), Axon (Union City, CA), BioDiscovery (Marina del Ray, CA), and many others make excellent products.
Recommended Equipment
NanoPrint 2 Protein Edition Microarrayers
946 Mioroarray Printing Technology
Stealth Microarray Printing Technology
SpotBot® 4 Protein Edition Personal Microarrayer
SuperEpoxy 3 Microarray Substrate Slides
Protein Printing Buffer
BlockIt Blocking Buffer
High Throughput Wash Station
Microarray High-Speed Centrifuge
Microarray Hybridization Cassette
Troubleshooting Tips
Poor printing quality:
- Incomplete mixing of protein samples in Protein Printing Buffer
- Poor printing environment (55% humidity and 25°C recommended).
- Clogged Micro Spotting Pins
Poor protein coupling:
- Poor surface chemistry (SuperEpoxy, SuperAldehyde or SuperAmine Substrates recommended)
- Inhibitor in protein sample (suspend in 1X PBS and then 1X PPB)
Weak fluorescent signals:
- Fluorescent protein probe mixture binds poorly to protein targets
- Probe labeling inefficient
- Washes too harsh
Ordering Information
Protein Printing Buffer, 50 ml of 2X solution, Cat. PPB
Warranty
Arrayit life sciences products are sold for research purposes only. 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 performance issues 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 is the full responsibility of the user, and Arrayit makes no warranty or guarantee under such circumstances. Pricing may vary up to 30% due to costs associated with distribution, import taxes, duties, customs clearance and shipping.