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Antibody Microarray

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					 Antibody Microarray
       User Manual
      Cat.# TAA-001




  Lab Vision Corporation
47790 Westinghouse Drive
   Fremont, CA 94539




      For Research Use Only
                                           Table of Contents
I.   INTRODUCTION
A.   Antibody Microarray                                          3
B.   Kit Components                                               4
C.   Storage Conditions and Handling Instructions                 4
D.   Reagents not provided with the Kit                           5

II. PROTOCOL
A. Protein Extraction                                             6
    A-1. Protein Extraction from Tissue                           6
    A-2. Protein Extraction from Cell Culture                     6
    A-3. Protein Extraction from Body Fluid                       7
B. Protein Labeling                                               7
    B-1. Biotinylation of the Protein Sample                      7
    B-2. Purification of Biotin Labeled Protein                   8
C. Hybridization of Labeled Proteins to the Microarray            8
D. Detection of Antibody Microarray                               9
E. Scanning                                                       10
F. Data Analysis                                                  11
G. Microarray Grid Structure                                      12
H. Protein Identification                                         12

III. OPTIONAL PROTOCOL
A. Protein Labeling                                               13
     A-1. Labeling Protein with Cy3 and Cy5                       13
     A-2. Purification of Labeled Protein                         14
     A-3. Dye/Protein Ratio Calculation                           14
B. Hybridization of Labeled Proteins to the Microarray            15
C. Washing and Drying Slides                                      16
D. Scanning                                                       16

IV. TROUBLESHOOTING GUIDE
A. High Background                                                18
B. Irregular Background                                           18
C. No or Low Signal Observed                                      19

V. APPENDIX
A. BUFFERS                                                        19
    1.     Blocking Buffer                                        19
    2.     Washing Buffer                                         19
B. FIGURES
    Figure 1. Schematic of Antibody Microarray Technology         20
    Figure 2. Schematic of the Microarray Slide                   21
    Figure 3. Mounting the chamber to the slides                  21
    Figure 4. Filling the chamber with labeled sample             21
    Figure 5. Microarray Layout                                   22
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I. INTRODUCTION

A. Antibody Microarray

   Microarray technology is far more prevalent today than it was a couple of decades ago when
   they were first introduced. Today DNA, RNA, peptides and proteins are available in array
   format and are being utilized by both biotech and pharmaceutical researchers, and in
   academia. Even though fewer antibody arrays are available compared to their nucleotide
   counterparts, the concept of screening for hundreds of proteins is rapidly catching on.
   Antibody microarrays have emerged as the tool of choice for high-throughput protein
   profiling, since the completion of the human genome project and the increasing role of
   protenomics in drug discovery and therapeutics. Microarray technology allows one to screen
   hundreds of samples simultaneously and therefore generate a large number of data points
   within a short period of time. Antibody microarrays are an easy-to-use, cost-effective tool
   for high-throughput protein profiling using cell extracts, tissue lysates, and other biological
   samples. The antibodies act as bait in binding proteins present in the biological samples,
   while the presence of the antibody-antigen interaction may be visualized by either
   Chemiluminescent or fluorescent signals.

   The arrays commercially available today vary in density, number of arrays per set, and
   medium used to immobilize the biological agents. The high arraying density of these
   microarrays offers tremendous advantages over traditional analytical methods, such as
   ELISA or Western blots. In addition to saving time, the microarray also minimizes the
   amount of reagents used conserving both the immobilized bait and the samples used. One of
   the inherent advantages of microarrays is that they introduce uniformity to the experiment,
   reducing the number of external variables that might influence experimental outcome. Using
   antibody microarrays, one can compare the expression patterns of multiple proteins in
   different tissues, different developmental stages, or different physiological states (normal
   versus diseased or treated versus untreated cells). Antibody microarrays have been
   successfully used to determine relative abundance of key proteins in various cancers and
   other diseases.

   The Lab Vision antibody microarray is the largest and most diverse antibody microarray to
   date among those offered by a relatively small group of companies. The Lab Vision antibody
   microarray consists of 720 high quality, well-characterized antibodies in triplicate, including
   several house keeping proteins as internal controls. Each standard size slide (dimensions: 75
   mm x 25 mm x 1 mm) consists of 2160 spots. These slides were carefully chosen to be
   compatible with most commercially available scanners. The microarray consists of many
   widely investigated research groups such as angiogenesis, apoptosis, signal transduction,
   transcriptional regulation, and neurobiology. The kit consists of two identical glass slides,
   (each containing 720 antibodies in triplicate), all reagents for protein extraction, protein
   labeling, hybridization, and detection, along with a comprehensive instruction manual. This
   format allows one to investigate relative abundance of a wider range of proteins, and
   compare protein abundance of the same proteins using different samples (e.g. diseased vs
   normal or treated vs untreated samples). The Lab Vision antibody Array adds value to the
   customer, as each antibody has been performance tested by WB, IP, and IHC applications.

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                                     For Research Use Only
    The microarrayed slides and key detection components are included in the kit. An optional
    Cy3/Cy5 direct labeling protocol is also included in section III of this handbook for the
    convenience of the end-user. This allows the end-user the flexibility of using various
    biological samples and using either fluorescent or chromogenic detection systems, but
    reagents supplied with this kit are specifically designed for the protocol included in this
    manual (illustrated in Figure 1, APPENDIX B). A complete list of what is included in this
    kit is given below.

B. Kit Components

Reagents/Materials           Qty                        Usage                 Storage
Antibody Array               2                          Microarray            4 oC
Extraction Buffer            2 ml                       Protein extraction    4 oC
Labeling Buffer              1 ml                       Protein labeling      4 oC
Biotinylation Reagent        1-2 mg                     Protein labeling      - 20 oC
Hybridization Buffer         1 ml                       Hybridization         4 oC
Streptavidin Solution        2 ml                       Detection             4 oC
Detection Antibody-Cy3       2 ml                       Detection             4 oC
Micro Spin Columns and
                             2                          Protein labeling      4 oC
Collection Tubes
Detection Chambers           4                          Detection             Room Temperature
Cover slips                  4                          Hybridization         Room Temperature
Microarray Description/      www.labvision.com
                                                        Data Analysis
User Manual                  or CD



C. Storage Conditions and Handling Instructions
   Upon receiving the antibody microarray kit, Biotination Reagent should be removed from the
   box and stored at -20 °C. Detection Chambers, and Cover slips should be stored at room
   temperature. All other components, including the slides, should be kept at 4 °C. When
   properly stored, all components in the kit are guaranteed for 12 months from the time the kit
   is received.

    Slides and chemicals should always be handled with gloves to avoid contamination from
    proteases, fluorescent, and bacterial materials. Glass slides are fragile and should be handled
    with plastic or flat-ended forceps. Never allow anything but solutions to touch the
    microarrayed area of the slides. The slide microarrayed area is illustrated in Figure 2,
    APPENDIX B.
.


D. Reagents not provided with the Kit
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                                      For Research Use Only
§   1x PBS
§   Tween 20
§   Blocking Buffer (3% BSA in 1x PBS, APPENDIX A)
§   Washing Buffer (APPENDIX A)
§   BCA protein assay kit (Pierce Cat # 23225)
§   BSA protein standard
§   Tissue homogenizer
§   N,N-Dimethylformamide (DMF)
§   Shaker
§   Compressed-air duster (oil-free) or swinging bucket centrifuge (with adaptors for spinning
    50ml tubes)
§   Micro centrifuge
§   Microarray slide scanner




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                                     For Research Use Only
II. PROTOCOL
    Note: Please read the manual carefully before use.

A. Protein Extraction

Any commonly used protein extraction method may be used to prepare the sample(s).

A-1. Protein Extraction from Tissue

   1. Transfer frozen tissue on to ice.

   2. Add enough Extraction Buffer to cover the tissue in a homogenizing tube.

   3. Homogenize the tissue on ice.

   4. Transfer the homogenate to a centrifugation tube (4 °C).

   5. Centrifuge the homogenate at 10,000 x g for 30 min at 4 °C.

   6. Transfer the supernatant to a clean tube.

   7. Measure protein concentration using BCA protein assay kit.

   8. Proceed immediately to Step B, Protein Labeling or store the samples at -80 °C.

A-2. Protein Extraction from Cells

   1. Harvest cells by centrifugation.

   2. Wash the cells with 1xPBS and spin at 4°C. Aspirate the supernatant.

   3. Wash the cells twice with cold 1x PBS and spin at 4 °C to completely remove serum
      proteins.

   4. Keep the cell pellet on ice for 5 min.

   5. Add Extraction Buffer to the cell pellet and mix thoroughly by pipetting.

   6. Incubate the sample on ice (4 °C) for 10 min with occasional mixing.


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   7. Centrifuge the sample at 10,000 x g for 30 min at 4 °C or in a micro-centrifuge at highest
      speed for 10 min at 4 °C.

   8. Transfer the supernatant to a clean tube.

   9. Measure protein concentration using BCA protein assay kit.

   10. Proceed immediately to Step B, Protein Labeling or store the samples at -80 °C.


A-3. Protein Extraction from body fluid

   1. Centrifuge the sample at 10,000 x g for 10 min or micro-centrifuge at highest speed for
      10 min at 4°C.

   2. Transfer the supernatant to a clean tube.

   3. Measure protein concentration using BCA protein assay kit.

   4. Proceed immediately to Step B, Protein Labeling or store the samples at -80 °C.


B. Protein Labeling

B-1.   Biotinylation of the Protein Sample

   1. Weigh 1 mg (or less) of Biotinylation Reagent and dissolve in 0.1 ml of DMF (N,N-
      Dimethylformamide) to make a solution of 10 mg/ml.

   2. Take 100µg or less of the protein sample (not to exceed 25µl) and dilute two fold with
      labeling buffer to a final volume of 50µl.

   3. Add 0.0143 mg of Biotinylation Reagent to 0.1 mg protein sample. The ratio of
      Biotinylation Reagent to protein sample should be maintained at 1:7 regardless of sample
      size.

   4. Mix and incubate at room temperature (23 – 25 °C) for 2 hrs with rotating or shaking or
      mixing.




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B-2.   Purification Biotin Labeled Proteins


   1. Invert the spin column several times to resuspend the gel.


   2. Snap off the tip and place the column in a 2-ml Collection tube.


   3. Remove the top cap and allow column to drain by gravity.


   4. Discard the buffer and return the column to the 2.0 ml Collection tube.


   5. Centrifuge for 2 minutes at 1,000 x g (In an Eppendorf micro centrifuge, spin at 4,000
      rpm for 2 minutes) to remove buffer from the column.


   6. Transfer column to a clean 1.5 ml micro centrifuge tube.


   7. Carefully apply the sample to the center of the column.


   8. Centrifuge the column for 4 minutes at 1,000 x g (In an Eppendorf micro centrifuge, spin
      at 4,000 rpm for 4 minutes) and collect the purified biotin labeled protein sample.



C. Hybridization of Biotin Labeled Proteins to the Microarray

   Note: Precautions must be taken when handling the antibody microarray slides, especially in
   making sure that nothing except the intended solutions touches the microarrayed area of the
   slides. It is essential to use protease-free water for preparing the necessary buffers.
   Hybridization should be performed at room temperature (23 – 25 °C). For identification
   purposes, the slides should be marked with water-resistant ink. Equal amounts of samples,
   prepared in parallel, under identical conditions should be used in all paired experiments (such
   as “diseased versus normal” or “treated versus untreated”).

   1. Block Antibody Microarray slides by soaking into Blocking Buffer (APPENDIX A) at
      room temperature for 30 min.

   2. Air-dry Antibody Microarray slides by placing the slides on paper towels.

   3. Add Hybridization Buffer to Biotin Labeled Protein Sample up to a total volume of 100µl.
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   4. Set Antibody Microarray slides in a humidity chamber or similar environment.

   5. Slowly put Cover slip (provided) on the slide and carefully apply the sample to the top of
      the microarrayed area (underneath the cover slip).

   6. Incubate at room temperature for 2 hrs (The incubation time could be up to 4hrs
      depending on the sensitivity of the system).

   7. Take the Antibody Microarray slides out of the humidity chamber and remove the Cover
      slip by dipping the slide into 50 ml of Washing Buffer (see APPENDIX A) in a 50-ml
      tube (Use caution not to scratch the microarrayed area).


D. Detection of Antibody Microarray

   1. Wash the slides with ~40 ml of Washing Buffer in a 50-ml tube with shaking for 10 min
      at room temperature. Wash twice more with Washing Buffer.

   2. Take the slides out of the tubes and lay on paper. Wipe out the moisture from the edges
      of the slides, keeping the microarrayed area wet. This step must be done carefully and
      quickly.

   3. Peel off the clear, thin plastic liner of the Detection chamber (provided).

   4. Carefully place the Detection chamber on the slide over the microarrayed area making
      sure that the chamber’s edges do not extend past the glass slide. Avoid applying strong
      pressure on the chamber during this step. Please see Figure 3 in APPENDIX B.

   5. Very slowly fill the Detection chamber through one hole of the chamber with 800 – 1000
      µl of Streptavidin Solution (provided) using a P200 pipette (recommended). Please see
      Figure 4 in APPENDIX B. Do not cover the holes during filling and incubation.
      Remove any air bubbles from the arrayed area of the slide.

   6. Place the slide on the bench top and incubate 45 min at room temperature (23 - 25 °C).

   7. Peel off the Detection chamber and rinse briefly by dipping the slide into a 50 ml conical
      tube filled with Washing Buffer (We recommend securing the slides in 50 ml conical
      tubes for the washing steps with 60 rpm constant shaking).

   8. Wash each slide separately in a 50 ml conical tube filled with ~35 ml of Washing Buffer
      at room temperature (23 - 25 °C) for 10 min., with 60 rpm constant shaking.


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     9. Change Washing Buffer and repeat Step 8 three additional times.

     10. Repeat steps D-2 through D-4, drying and applying the second detection chamber to the
         slide.

     11. Very slowly fill the Detection chamber through one hole of the chamber with 800 – 1000
         µl of Detection Antibody-Cy3 (provided) using a P200 pipette (recommended). Please
         see Figure 4 in APPENDIX B. Do not cover the holes during filling and incubation.
         Remove any air bubbles from the arrayed area of the slide.

     12. Place the slide on the bench top and incubate 45 min at room temperature (23 - 25 °C).

     13. Peel off the Detection chamber and rinse briefly by dipping the slide into a 50 ml conical
         tube filled with Washing Buffer (We recommend securing the slides in 50 ml conical
         tubes for the washing steps with 60 rpm constant shaking).

     14. Wash each slide separately, with 60 rpm constant shaking, in a 50 ml conical tube filled
         with ~35 ml of Washing Buffer at room temperature (23 - 25 °C) for 10 min.

     15. Change Washing Buffer and repeat Step 14 three additional times.

     16. Rinse by quickly dipping the slides in a 50 ml conical tube filled with clean water. Dry
         with an oil-free, compressed-air duster or by centrifugation at 1000 x g for 1 min. (Note:
         use a rotor with swinging buckets).

     17. Check to make sure that no salt residue is left on the surface of the slides otherwise repeat
         step D-16.

     18. The slides are now ready for scanning.


E. Scanning

1.   Slide Storage after Hybridization
     When the slides are protected from light in a dry area, the signal intensity is stable for several
     weeks. For storage, use the plastic slide box supplied with the kit and wrap in aluminum foil.

2. Cy3 Spectral Characteristics
   The Detection Reagent is conjugated to cyanine-3 fluorescent dyes (Cy3), which emits light
   when stimulated by an appropriate excitation wavelength.



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                                        For Research Use Only
The spectral characteristic of Cy3 is as follows:
                                            Cy3
   Excitation wavelength:                      550 nm
   Emission wavelength:                        570 nm
   Color of fluorescence:                      Orange Red

3. Scanning Hardware
   Completely processed and dried antibody microarray slides are ready to be scanned.
   Scanning may be performed with most commercial laser scanners, equipped with laser
   source and filters compatible with the spectral characteristics of Cy3. Examples are:

   Affymetrix 417-418 (Affymetrix / Genetic MicroSystems) www.affymetrix.com
   ScanMicroarray Series (GSI Lumonics)             www.gsilumonics.com
   GenePix 4000A (Axon Instruments)              www.axon.com
   ChipReader (Virtex)                    www.virtex.ca

   Please note that by listing these scanners Lab Vision Corporation is not endorsing or
   recommending any of these brands over other commercially available scanners. It is the
   responsibility of the user to determine which scanner is appropriate for their lab setting by
   contacting the scanner’s manufacturer/distributor.

4. Scanning
   Insert a slide in the scanning device so that the microarray is facing the laser beam. Adjust
   the laser, channel and photomultiplier (PMT) setting to obtain the desired sensitivity and
   detection wavelength. Scan the microarray at the highest PMT and laser settings that
   preserve linearity and minimize background. Follow scanner manufacturer’s instructions to
   optimize scanning.


F. Data Analysis

   1. Hardware and Software
   To quantify the fluorescence emission from each spot of the microarray, use a microarray
   image analysis software such as ImaGene from BioDiscovery Inc. (www.biodiscovery.com),
   or those from GSI Lumonics, Axon, or Silicon Genetics. The minimum hardware
   specifications for running this type of software is a computer with a 100 MHz Pentium
   processor and 64 MB RAM, although the programs run better on a computer with a faster
   processor and more RAM.

   2. Data Processing
   With image analysis software, the fluorescence signals from two samples can be easily
   processed for differential expression analysis. For instance, protein levels in one tissue can be

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   compared with that in another tissue. The ratio of fluorescent intensities for a spot is
   interpreted as a ratio of concentration for the corresponding protein in the two tissues.

   The relative expression level of each protein can be determined by analyzing the signal
   intensity on each dot after normalizing with the total intensity of Cy3. For differential
   expression analysis between 2 slides, the house keeping proteins can also be used to
   normalize the sample-to-sample variations; however normalizing to the entire population of
   spots is preferred in most cases.

   Though current commercial data image analysis software can do a great job of converting the
   scanned image information into numeric data, some difficulties might occur when overlaying
   the images from two different samples. In such instances, it is recommended to analyze each
   sample separately, and export coordinates and raw data into spreadsheet software such as
   Microsoft Excel. Simple formulas can be applied to the raw data to normalize, calculate
   expression ratios, filter the information and draw simple scatter plots or histograms.
   Thresholds can be applied to eliminate meaningless information generated by spots with
   intensities close to background levels. Also, protein/antibody information can be added to
   the spreadsheet.


G. Microarray Grid Structure

   Antibody microarray consists of spots arranged into grids. The Lab Vision Antibody
   microarray has 16 sub-grids of identical dimension. The sub-grid structure is a microarray
   manufacturing artifact due to the use of printing heads with 8 spotting pins. Please see
   Figure 5 in APPENDIX B. The companion Compact disk (CD) contains an Excel file that
   can be used to sort the gene list according to different combinations of scanning devices and
   data analysis software.


H. Protein Identification

   A list of the proteins/antibodies represented in each of our slides is provided in the
   companion CD. Recent updates can also be downloaded from our website
   www.labvision.com. In addition to providing the most updated product information, Lab
   Vision’s website also allows you to search and retrieve information of protein categories
   represented on all of our microarrays.




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III. OPTIONAL PROTOCOL:

Reagents not provided with the Kit

§   1x PBS
§   Tween 20
§   Blocking Buffer (3% BSA in 1x PBS, See APPENDIX A)
§   Washing Buffer (1x PBST, See APPENDIX A)
§   BCA protein assay kit (Pierce Cat # 23225)
§   BSA protein standard
§   Cy3 mono-reactive dye pack (Amersham, Cat# PA23001)
§   Cy5 mono-reactive dye pack (Amersham, Cat# PA25001)
§   Tissue homogenizer
§   Shaker
§   Compressed-air duster (oil-free) or swinging bucket centrifuge (with adaptors for spinning
    50ml tubes)
§   Micro centrifuge
§   Spectrometer capable of measuring absorbance at 552 nm and 650 nm
§   Microarray slide scanner


A. Protein Labeling

A-1.   Labeling Protein with Cy3 and Cy5

    1. Take 40 µl of the sample containing 10 – 100 µg protein from Protein Extraction II A-1
       Step 8, or A-2 Step 10, or A-3 Step 4. Label one tube as control and one as sample.

    2. Resuspend Cy3 and Cy5 dyes (from Amersham) in 45 µl DMSO each.

    3. Add 5 µl Cy3 and Cy5 dyes into the control tube and the sample tubes respectively.

    4. Add 5 µl Labeling Buffer in each tube.

    5. Mix thoroughly with pipette (prevent foaming). Cap the vials to prevent light from
       penetrating into the mix.

    6. Incubate at 4 °C for 2 hours with additional mixing every 10 min.

    7. Incubate at room temperature (23 – 25 °C) for additional 20 min.

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A-2.   Purification of Labeled Proteins

   1. Invert the spin column several times to resuspend the gel.


   2. Snap off the tip and place the column in a 2-ml Collection tube.


   3. Remove the top cap and allow column to drain by gravity.


   4. Discard the buffer and return the column to the 2.0 ml Collection tube.


   5. Centrifuge for 2 minutes at1,000 x g (In an Eppendof micro centrifuge, spin at 4,000 rpm
      for 2 minutes) to remove buffer from the column.


   6. Transfer column to a clean 1.5 ml micro centrifuge tube.


   7. Carefully apply the sample to the center of the column.


   8. Centrifuge the column for 4 minutes at 1,000 x g (In an Eppendof micro centrifuge, spin
      at 4,000 rpm for 4 minutes) and collect the purified labeled protein sample. The collected
      volume from one column is 30 – 40 µl.



A-3.   Dye/Protein Ratio Calculation

For Cy3/protein Ratio

   1. Dilute a portion of the labeled sample so that the maximum absorbance is 0.5 to 1.5 AU.

   2. Read the diluted sample at 552 nm for Cy3 dye to obtain the absorbance at A552.

   3. [Cy3 dye] = A552/ 1.5 x 105

   4. Read the diluted sample at 280 nm to obtain the absorbance at A280.

   5. [Labeled protein] = [A280 – (0.08 x A552)]/1.7 x 105



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   6. The Cy3/Protein ratio = [Cy3 dye]/ [Labeled protein] = A552/1.5 x 105/
      [A280 - (0.08 x A552)]/ 1.7 x 105

   7. A ratio of 2 – 4 is acceptable, 2 being ideal. Note: A ratio of less than 1.0 will affect the
      sensitivity of the assay, while a ratio of greater than 6 will interfere with antibody binding.


For Cy5/protein Ratio

   1. Dilute a portion of the labeled sample so that the maximum absorbance is 0.5 to 1.5 AU.

   2. Read the diluted sample at 650 nm for Cy5 dye to obtain the absorbance at A650.

   3. [Cy5 dye] = A650/ 2.5 x105

   4. Read the diluted sample at 280 nm to obtain the absorbance at A280.

   5. [Labeled protein] = [A280 – (0.05 x A650)]/ 1.7 x 105

   6. The Cy5/Protein ratio = [Cy5 dye]/ [Labeled protein] = A650/ 2.5 x 105/ [A280 – (0.05 x
      A650)]/ 1.7x 105.

   7. A ratio of 2 – 4 is acceptable, 2 being ideal. Note: A ratio of less than 1.0 will affect the
      sensitivity of the assay, while a ratio of greater than 6 will interfere with antibody binding.


B. Hybridization of Labeled Proteins to the Microarray

   Note: Precautions must be taken when handling the antibody microarray slides, especially in
   making sure that nothing except the intended solutions touches the microarrayed area of the
   slides. It is essential to use protease-free water for preparing the necessary buffers.
   Hybridization should be performed at room temperature (23 – 25 °C). For identification
   purposes, the slides should be marked with water-resistant ink. Equal amounts of samples,
   prepared in parallel, under identical conditions should be used in all paired experiments (such
   as “diseased versus normal” or “treated versus untreated”).

   1.    Block Antibody Microarray slides by soaking into Blocking Buffer (APPENDIX A) at
        room temperature for 30 min.

   2. Briefly dry Antibody Microarray slides by standing on paper.



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     3. Mix 15 µl labeled protein from the control and the sample (A-2 Step 8). The total volume
        is 30 µl.

     4. Add 30 µl Hybridization Buffer to Labeled Protein.

     5. Set Antibody Microarray slides in a humidity chamber or any humid environment.

     6. Carefully apply the sample by dropping to the top of the microarrayed area and slowly
        put Cover slip (provided) on top of the sample.

     7. Incubate at room temperature for 2 hrs (The incubation can be up to 4 hrs, if sensitivity is
        a problem).

     8. Take Antibody Microarray slides out of humidity chamber and remove the Cover slip by
        dipping the slide into 50 ml of Washing Buffer (see APPENDIX A) in a 50-ml tube (Do
        not scratch the microarrayed area).


C. Washing and Drying of Slides

     1. Wash the slide with ~40 ml of Washing Buffer in a 50-ml tube with shaking for 10 min at
        room temperature. Wash three additional times with Washing Buffer.

     2. Rinse by quickly dipping the slides in a 50 ml conical tube filled with clean water. Dry
        with an oil-free, compressed-air duster or by centrifugation at 1000 x g for 1 min. (Note:
        use a rotor with swinging buckets).

     3. Check to make sure that no salt residue is left on the surface of the slides; otherwise
        repeat the rinsing.

     4. The slides are now ready for scanning. See section II. E for details.


D. Scanning

1.   Slide Storage after Hybridization
     When the slides are protected from light in a dry area, the signal intensity is stable for several
     weeks. For storage, use the plastic slide box supplied with the kit and wrap in aluminum foil.

2. Cy3/Cy5 Spectral Characteristics
   The Detection Reagent is conjugated to cyanine-3 fluorescent dyes (Cy3) and cyanine-5
   fluorescent dyes (Cy5), which emit light when stimulated by an appropriate excitation
   wavelength.
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The spectral characteristic of Cy3/Cy5 is as follows:

                                      Cy3                 Cy5
   Excitation wavelength:             550 nm              649 nm
   Emission wavelength:               570 nm              670 nm




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III.      TROUBLESHOOTING GUIDE

A. High Background

       High background is frequently the result of inappropriate blocking or washing conditions.
       Blocking with 3% BSA in 1x PBS is critical for reducing the amount of non-specific
       background. Thorough washing is also required to keep background levels low.

1. Dry slides: Avoid letting the slides to dry between steps as background levels will increase
   greatly. Slides should be transferred between solutions quickly in order to avoid drying.

2. Blocking: Block the slides before hybridization. Increasing blocking time may help reduce
   nonspecific binding to the slide.

3. Washing Conditions: Moderate increases in the stringency of the washing steps can
   significantly reduce background levels without changing the signal strength significantly.
   Increased temperature, time, or Tween 20 in the Washing Buffer will help reduce the
   background. After hybridization, increasing the washing temperature will help reduce non–
   specific binding of proteins. Wash time may also be increased in 5 min. increments until
   desirable results are achieved. Tween 20 in Washing Buffer can be increased up to 0.1%.


B. Irregular Background

       Occasionally, background may appear spotty, blotchy, or uneven. This may be caused by one
       of the following: scratches slides, bubbles trapped during hybridization, or poor mixing of
       solutions.

1. Scratched Slides: Handle the slides with the utmost care throughout the procedure,
   especially when placing or removing the Cover slip. Also use caution to avoid any direct
   contact with the microarrayed area to prevent irreversible damage.

2. Hybridization: It is important to take all necessary precautions noted in the procedure
   during the hybridization steps. Improper placement of the cover slip will cause leakage and
   may result in evaporation or shifting of the Cover slip. This can damage the microarray,
   creating uneven signal distribution and irregular or high background. When filling the sample,
   make sure that all bubbles are moved to the edge of the slide. A total of 100 µl should be
   used to cover the microarrayed area underneath the cover slip.

3. Wash Steps: Follow the simple procedure described in the protocol. Make sure that the
   washing steps are performed in a volume sufficient to cover the microarray completely at all
   times to avoid irregular background or signals. The wash steps should be done with constant
   shaking at low speed (~ 60 rpm) at 23-25 °C.
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C. No or Low Signal Observed

   Lack of signal may be a result of too little protein, inefficient labeling of the protein, or
   improper hybridization and washing.

   1. Amount of Protein: If the labeled protein does not yield a strong enough signal, it may
      be necessary to increase the total amount of labeled protein.

   2. Labeling Ratio: Check the labeling ratio as described in the manual.

   3. Hybridization and Washing: Carefully follow the manual for hybridization and wash
      steps. Make sure to prevent leaking of the hybridization buffer from the chamber. Wash
      the slides gently.

   4. If a product is found defective, please notify our Customer Service Department promptly
      by calling 800-828-1628, so that we can determine the nature of the problem and remedy
      it.




IV. APPENDIX

A: BUFFERS

   1. Blocking Buffer: 3% BSA in 1x PBS

   2. Washing Buffer: 1x PBS containing 0.05% Tween 20




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                                       For Research Use Only
B: FIGURES

                       Sample A                 Sample B
                                     Protein extraction
                                          (30min)


                                      Protein Labeling
                                          (2-3hrs)



                                   Remove unbound dye
                                        (30min)




                                        Hybridization
                                          (2-4 hrs)




                                        Detection (2hrs)



                                      Scan / Data Analysis



Figure 1: Schematic of Antibody Microarray Technology




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                                  For Research Use Only
Figure 2. Schematic of the Arrayed Slide:




Figure 3. Mount the chamber on the slide over the microarrayed area




Figure 4. Fill the Detection chamber with the Streptavidin or Antibody Solution
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                                   For Research Use Only
Figure 5. Antibody Microarray Layout




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                                For Research Use Only