Microarray Buffers - Blocking Solutions - BlockIt™ and BlockIt™ HT Blocking Buffers for DNA and Protein Microarrays

Arrayit has developed and manufactured an advanced microarray blocking solution designed to inactivate reactive groups remaining post-printing on SuperEpoxy 3, SuperAmine 3, SuperAldehyde 3 and other glass microarray substrate slide surfaces. BlockIt™ Blocking Solution greatly reduces background noise while maintaining full signal intensities for a wide spectrum of DNA and protein microarray applications. 250 ml of 1X buffer.

Table of Contents

• Introduction
• Quality Control
• Product Description
• Technical Assistance
• Short Protocol
• Complete Protocol
• Short Protocol DNA
• Complete Protocol DNA
• Troubleshooting Tips
• Recommended Requirements
• Ordering Information
• Warranty

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your genomics research. This booklet contains a complete set of protocols outlining the steps and principles needed to use the Arrayit brand BlockIt blocking buffer for microarray surface chemistries. This product is design for use prior to incubation and hybridization reactions involving protein and DNA microarrays.

Quality Control
Arrayit assures the performance of these products, the finest scientific research went into their development. Store at 4°C, the product has a 1-year shelf life when handled and stored properly.

Product Description
Arrayit brand BlockIt™ buffer is designed for blocking the portions of microarrays that are still reactive after printing to eliminate fluorescent background following the incubation or hybridization reaction of protein and DNA microarrays. Users will appreciate the following features:

• Ultra-pure reagents used for buffer formulation
• Purified by sterile filtration
• Provided as 1X ready to use concentrate
• Recommended for use on all types of protein or DNA microarray glass microarray surface chemistries
• Easy cover slip blocking procedure for minimal reagent consumption
• 250 ml volume provided efficiently blocks thousands of microarrays
• Will not cross react with proteins and DNA

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 Arrayit technical assistance). By email: arrayit@arrayit.com, Monday–Friday PST 8:00am - 8:00pm. Please remember that we want to hear about your successes!

Short Protocol for Protein Microarrays
Wash microarray and then incubate microarray in 1X BlockIt buffer under a coverslip for 1 hour prior to protein binding or hybridization reactions.

Complete Protocol for Protein Microarrays
BlockIt will couple to un-reacted binding groups on microarray surfaces and prevent background fluorescence. Once the printing process is complete, and samples are coupled to the surface, block a microarray surface by using a 1-24 hour incubation at room temperature in 1X BlockIt buffer. Perform this reaction under a coverslip using 4 ul of BlockIt for every cm2 of coverslip area. A Hybridization Cassette should be used to keep the blocking reaction from drying out. After blocking, wash the microarrays to remove excess buffer. Wash 2 times for 15 minutes each in a High Throughput Wash Station in 1X PBST to remove unbound protein molecules and buffer components from the microarray. Protein and DNA binding to the SuperEpoxy 3 surface is extremely stable and the microarrays can be washed, blocked and reacted without sufficient loss of coupled protein.

NOTE: BlockIt can be used as a reaction buffer for all protein microarray applications.

Figure 1. BlockIt™ background fluorescence reduction on SuperAmine (left panel) and SuperEpoxy (right panel) microarray glass substrate slides. Blocking reactions were performed at room temperature for 16 hours. 10 µl of BlockIt™ was used under 22 x 22 mm coverslips. The substrate slides were washed in 1x PBS in a High Throughput Wash Station for 10 minutes at medium speed on a stir plate. The substrate was spun in a Microarray High-Speed Centrifuge until dry. A total of 30 µl of 0.6% Cy3-labeled IgG was reacted across the substrate slides under 20 x 60 mm coverslips. The IgG was suspended in a 1% BSA in PBS solution. The solution was allowed to react for 4 hours at room temperature protected from light. The substrate slides were washed in 1x PBS in a High Throughput Wash Station for 10 minutes and spun dry. The substrate slides were scanned using a PerkinElmer ScanArray Express set at 70% PMT and 90% laser power at a 10 µm resolution. The signal intensities (see Table) were averaged across a circular area with an 8,575 µm diameter. The upper panel shows the scanned images and the table records the signal intensities. BlockIt™ blocking solution reduces background noise by 2-10 fold depending on the surface.

Short Protocol for DNA Microarrays
Incubate microarray in 1X BlockIt™ buffer under a coverslip or in a microarray reaction tray for 15-60 min prior to hybridization.

Complete Protocol for DNA Microarrays
BlockIt will couple to the un-reacted groups on the surfaces and prevent background fluorescence. Once the printing process is complete, and samples are bound to the surface use a 1-24 hour incubation at room temperature of 1X BlockIt buffer. Perform this reaction under a cover slip using 4 ul of BlockIt for every cm2 of coverslip area. A Hybridization Cassette can be used to keep the blocking reaction from drying out. After blocking wash the printed microarrays in a High Throughput Wash Station using the protocol recommended by the manufacturer. DNA binding to the SuperAmine 3, SuperAldehyde 3 and SuperEpoxy 3 surfaces is extremely stable and the microarrays can be washed, blocked and reacted without significant loss of coupled DNA. After blocking, wash and process the microarrays according to the manufacturers recommendation to remove excess buffer and unbound sample.

Figure 2. Shown is a microarray blocking reaction using BlockIt™ Microarray Blocking Buffer.  Three DNA microarrays are incubated overnight with gentle agitation in a square culture dish containing 30 ml of 1X BlockIt™ to inactivate the microarray surface and reduce fluorescent background during the hybridization reaction. BlockIt™ molecules bind covalently to un-reacted groups on the microarray surface, preventing non-specific binding of probe molecules during hybridization. Unlike other blocking solutions that contain cloudy additives and impure reagents, BlockIt™ is perfectly clear solution of highly purified and soluble components that provides a vastly superior blocking function.  BlockIt™ is an all-purpose blocking solution for DNA, protein, and other types of microarrays.

Troubleshooting Tips
If microarray shows high background:

• Confirm that the intrinsic background of the microarray is low to start with.
• Be sure to not let any sample dry on the microarray surface during the binding reaction.
• Make sure to purify away unincorporated dyes in the labeling reaction.
• Take care to block for the recommended time (1 hour).
• Pay attention to the proper handling and shelf life of the buffer
  (1 year shelf life, Store at 4 C).
• Do not skip washing the microarray after the blocking step.

Recommended Products:

• Hybridization Cassette or Hybridization Cassette Plus
• High Throughput Wash Station
• Microarray Centrifuge