Scanning the Antibody Array

Each sub-array contains two spots with a monoclonal antibody specific for Cy3/Cy5, which serve as positive controls. These points can be used as internal references for positioning the sub-arrays. Furthermore, the array is spotted with buffer at several spots in order to serve as controls for non-specific signals. Refer to file “Antibody List, Specificity & Position” on the accompanying USB memory stick or at www.sigma.com/arrays.

The slides should be absolutely dry before the scanning procedure. Water may cause background problems.

Due to the short half-life of the dyes it is recommended that the array be scanned as soon as the experiment is completed and no more than 24–48 hours later.

The arrays can be scanned with most commercially available ‘overhead’ light source DNA array scanners that accommodate standard microscope slides. Scanners with bottom-lit light sources (e.g., Agilent) cannot be used, as the nitrocellulose coating employed with antibody arrays will interfere with the instrument’s signal detection system. The optimal laser power and PMT should be determined for each scanner individually.

Instrument Compatibility:

  • Accommodation of a standard microscope glass slide (25 mm x 75.6 mm x 1 mm).
  • Light filter system reading within the near-red spectrum of cyanine dyes:

        Cy3
        excitation 550 nm
        emission 570 nm

        Cy5
        excitation 659 nm
        emission 670 nm

Typical Scanner Settings:
The optimal laser power and PMT (Photomultiplier Tube) parameters should be determined for each scanner individually. The following parameters are a recommendation in order to obtain a signal-noise ratio greater than 10:1.

            Laser power: 40–80%
            PMT: 40–80%

 

  • It is advised to use the lower settings for the initial scan. Depending on the abundance of protein and thus, the corresponding spot signal intensity, the highest settings may accentuate the nitrocellulose background, thereby, “washing-out” any positive bound-protein signals.
  • Confocal plane focus should be adjusted for arrays from different batches. Nitrocellulose coating thickness may vary between 7–10 µm.

Use the attached Excel worksheet file found on the USB memory stick for compiling these results.

Note: Occasionally, positive control spots may vary in intensity, resulting in low to high responses.

Materials

     
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