Alkaline Phosphatase Labeling Kit, 100 Reactions

Arrayit Alkaline Phosphatase Labeling Kit, 100 Reactions provides a premium solutions for DNA and protein microarray-based alkaline phosphatase (AP) labeling and detection. This innovative hardware and reagent kit makes alkaline phosphatase assays rapid, affordable and easy to implement. Arrayit APK contains AP developing reagents and reaction cassettes for developing microarrays containing alkaline phosphatase-linked labeling reagents. The bright blue colorimetric microarray chemistry increases reagent and pigment stability and improves assay detectivity. Light-proof bottles prevent photo-degradation, 30 min reaction times, 60 min total assay time, 1,000-fold dynamic range, for glass and membrane-coated slides, 100 AP reactions per kit, ships on cold packs, 1 kit per order.

Amplification and Labeling - Alkaline Phosphatase (AP) Kits for DNA and Protein Microarrays in Research, Life Sciences, Microarray Testing and Pharmaceuticals

Arrayit Alkaline Phosphatase Labeling Kits enable rapid and afforable DNA and protein microarray research and life sciences based on alkaline phosphatase labeling and colorimetric detection. Arrayit sets the standard for innovative colorimetric microarray solutions by offering APK, which allows customers to process 100 microarray reactions per kit, with 30 min development times, 60 min total assays times, 1,000-fold dynamic range and bright blue signals for easy colorimetric detection of microarray spots. Miniaturize your favorite assay today!

Table of Contents

• Introduction
• Quality Control
• Product Description
• Kit Contents
• Short Protocol
• Complete Protocol
• Troubleshooting Tips
• Recommended Products
• Technical Support
• Scientific Publications
• Ordering Information
• Warranty

Introduction
Congratulations on taking a big step towards improving your DNA and protein microarray research. This booklet contains complete protocols outlining the steps required to use the Arrayit Alkaline Phosphatase Kits.

Quality Control
Arrayit assures the performance of this product line. The highest levels of scientific research and innovation were used to develop these kits.

Product Description
Arrayit provides premium kits for alkaline phosphatase (AP)-based microarray research and testing. This innovative hardware and assay solution makes alkaline phosphatase assays rapid, affordable and easy to implement. Users will appreciate the following features:

• Contains AP developing reagents and assay hardware
• Novel blue colorimetric chemistry increases reagent and pigment stability
• Light-proof bottles prevent photo-degradation
• Develops any alkaline phosphatase microarray
• Use on membrane-coated and glass slide surfaces
• Rapid reaction time of 1-30 min
• Total assay time of 60 min
• 1,000-fold dynamic range
• High assay sensitivity and low background
• Affordable at pennies per microarray spot
• Hardware can also be used for upstream protein and antibody binding steps
• Convert any conventional AP assay into a microarray format
• Environmentally friendly formulation contains no organic solvents
• 100 reactions per kit

Kit Contents
Arrayit Alkaline Phosphatase Kits (Cat. APK) contain the following components:

• Five dual substrate slide (1 x 25 x 76 mm) reaction cassettes
• Five glass cover slips (24 x 60 mm)
• One bottle of AP Reaction Buffer (30 ml of 3X solution)
• One bottle of 3X AP Color A (30 ml of 3X solution)
• One bottle of 3X AP Color B (30 ml of 3X solution)

Short Protocol (steps 1-7)
1. Place alkaline phosphatase microarray into reaction cassette.
2. Prepare 300 µl of AP Developer.
3. Transfer 125 µl AP Developer onto the microarray.
4. Add cover slip and incubate 1-30 min at RT.
5. Stop the reaction by flooding cassette with dH2O.
6. Spin dry by centrifugation.
7. Scan microarray to detect blue colorimetric signals.

Complete Protocol (steps 1-7)
1. Place alkaline phosphatase microarray into reaction cassette. Prepare any microarray slide (Arrayit SuperNitro Microarray Substrate Slides, Cat. SMN shown here) containing a linked alkaline phosphatase reagent such as an antibody-AP conjugate. Place the two microarray slides with the microarray spots facing upward in the bottom portion of the reaction cassette (Fig. 1). Make sure the glass slides are seated firmly against the cassette base.

Figure 1.  Placing two microarray slides (Arrayit SuperNitro, 1 x 25 x 76 mm) into the base of the alkaline phosphatase reaction cassette. The cassette lid snaps closed to prevent evaporation during the AP development step.

2. Prepare 300 µl of AP Developer. Thaw all three bottles of reagents, which should be stored frozen at –20°C before and after use. After thawing, mix the reagents thoroughly before use by inverting and swirling the bottles 5-10 times with the caps tightly sealed. Failure the mix the reagents at this step will reduce assay performance. To a microfuge tube, add 100 µl of 3X AP Reaction Buffer, 100 µl of 3X AP Color A, and 100 µl of 3X AP Color B, and mix thoroughly. The 300-µl mixture of AP developer is sufficient to develop 2 microarray slides at 125 µl per microarray slide under the 24 x 60 mm cover slips.

3. Transfer 125 µl AP Developer onto the microarray slide. Using a 200-µl pipette, pipette 125 µl of AP Developer from the 300 µl mixture and place the 125-µl droplet directly onto the microarray surface (Fig. 2). Move quickly to Step 4 to avoid uneven AP developing.

Figure 2. Pipetting 125 µl of AP developer onto the microarray surface. Arrayit SuperNitro Microarray Substrate Slides (Cat. SMN) are shown here. Note that for proper orientation, the corner chamfer of the microarray slide should be located in the upper right corner of the slide as shown. Move quickly to place the glass cover slip resting on the cassette lid (left) onto the AP developer droplet (see Fig. 3) to avoid uneven developing.

4. Add cover slip and incubate 1-30 min at RT. After pipetting the 125-µl drop of AP developer onto the microarray slide, use fine forceps to immediately place a 24 x 60 mm cover slip directly onto the droplet. Use fine forceps to lower the cover slip slowly (Fig. 3). Care should be taken not to introduce air bubbles under the cover slip. The AP developer should sheet evenly across the entire area of the cover slip, forming a uniform 87-µm layer of reagent across the microarray. This reagent thickness provides a sufficient reagent concentration to develop any type of alkaline phosphatase microarray. The AP developer reaction can be incubated at room temperature (20-25°C) or at 37°C for accelerated reaction kinetics. The total incubation time is 1-30 min depending on the amount of AP enzyme on each microarray. One approach is to incubate one microarray for 1-5 min and a second microarray for 20-30 min, to obtain different “exposures” spanning a wide range of signal intensities corresponding to weak and strong signals. Some optimization may be required to determine the correct development time. Excessive development time will lead to a loss of quantitation and elevated background, and should be avoided.

Figure 3. Placing a 24 x 60 mm glass cover slip onto a 125-µl droplet of AP developing reagent. Place the cover slip onto the slide slowly to allow the AP developing reagent to sheet across the entire microarray slowly and uniformly. Arrayit SuperNitro Microarray Substrate Slides (Cat. SMN) are shown here.

5. Stop the reaction by flooding cassette with dH2O. After 1-30 min of developing time, carefully open the reaction cassette, secure the microarray slides in position by applying light pressure between thumb and forefingers, and flood the surface of the microarray slides with a steady stream of distilled H2O (Fig. 4). The glass cover slips should float free and fall off of the microarray slides at this step. Make sure the cover slip dislodges within the first 10 seconds of washing to prevent uneven developing. The AP development reaction will be stopped completely after 30 seconds of washing. Close the cassette lid and flip it downward several times by hand to remove residual dH2O (Fig. 5) to provide nearly dry microarrays that are ready for complete drying by centrifugation.

Figure 4. Stopping the alkaline phosphatase development reaction using a steady stream of distilled water. This step removes AP developing reagent from the microarray surface and prevents over-development, while keeping the microarray spots and signals intact. Arrayit SuperNitro Microarray Substrate Slides (Cat. SMN) are shown here.

Figure 5.  Removing residual distilled water after the wash step of the alkaline phosphatase protocol.  The cover slips should be removed from both microarrays and the reaction cassette should be closed completely before flipping the cassette downward to remove the dH20. Arrayit SuperNitro Microarray Substrate Slides (Cat. SMN) are shown here.

6. Spin dry by centrifugation. Wearing safety glasses as a safety precaution, place the microarray in a Microarray High Speed Centrifuge, close the centrifuge lid, and spin for 10 seconds to dry the microarray slide completely (Figure 6). Repeat with the second microarray slide and all others until each is dry. The dry microarray slides are now ready for colorimetric scanning. The blue colorimetric microarray spots can also be inspected visually using a dissecting microscope (20-40X power).

Figure 6. Drying an alkaline phosphatase microarray slide in a Microarray High Speed Centrifuge (Cat. MHC110V). Make sure the microarray slide is inserted completely into the plastic substrate slide holder before commencing centrifugation. Wear safety glasses at all times when operating this instrument.

7. Scan microarray to detect blue colorimetric signals. Use the Colorimetric Microarray Scanner to detect the blue AP signals and acquire a quantifiable TIFF image. Place one or more microarray slides with the spots facing downward in the scanner template towards the scanner. In the proper orientation, the white membrane will be facing downwards and the corner chamfer will be in the upper left (Fig. 7). Use the SpotWareTM controller software to select scan area, resolution, gain, and other scanning parameters. A 16-bit TIFF file containing the AP data is now ready for quantification and modeling.

Figure 7. Developed alkaline phosphatase microarray slide scan-ready in a Colorimetric Microarray Scanner. The Arrayit nitrocellulose slide (Cat. SMN) with colorimetric signals is shown in template position 4 on the bed of the SpotWare Colorimetric Scanner with the pigmented microarray spots facing downward. SpotWare scanner software is used to select scan area, scan resolution, scan gain and other parameters.

Troubleshooting Tips
Low signal

• Increase development time
• Use Arrayit SuperProtein Blocking Buffer (Cat. SPBB) for blocking
• Increase scan gain

High background

• Reduce development time
• Reduce scan gain
• Block microarray slides with Arrayit Blocking Solutions

Recommended Products
Arrayit microarray printers
Arrayit colorimetric microarray scanners
Arrayit SuperProtein Microarray Substrate Slides
Arrayit SuperNitro Microarray Substrate Slides
Arrayit SuperEpoxy Microarray Substrate Slides
Arrayit Protein Printing Buffer