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Sample Incubation on the Array

Before starting the array assay, make sure that the dye to protein molar ratio (D/P) is > 2. A lower ratio may work; however, it may cause background problems.

The incubation time for the assay is short. When the signal is low, it is recommended to prolong the incubation time, but not longer than 45 minutes.

The control and test samples are labeled with different dyes and are mixed before applying on the array. These samples may have different D/P values. It is recommended to mix equal amounts of protein from the two different samples rather than equal dye concentration. The results can then be normalized according to the D/P ratio of each sample (control and test). To verify results, each control and test sample should be labeled by both Cy3 and Cy5 and mixed with its counterpart labeled with the other dye. Thus the experiment is fully controlled and doubly tested.

Note: Absolutely do not touch the surface of the array during the whole procedure. Forceps are recommended.

The incubation procedure is performed at room temperature.

  1. Mark each slide using only a pencil. A pen or a marker may affect the final results.
  2. Wash each slide briefly by dipping in PBS.
  3. In a tube add the Cy3 and Cy5 labeled samples at equal protein concentrations (~20 µg/mL each) to 5 mL of Array Incubation Buffer (Product No. A9602). Mix well by inverting the tube. Do not vortex.
  4. Add the mixture to well 1 of the incubation tray (QuadriPREM Cell Culture Vessel) supplied in the kit.
  5. Dip the slide into well 1 that already contains the labeled samples. Cover with the lid. Protect the plate from exposure to light by covering with aluminum foil.
  6. Incubate for 35-45 minutes at room temperature on a rocking shaker at a moderate shaking frequency of approximately 30 rpm.
  7. Add 5 mL Washing buffer to wells 2, 3 and 4.
  8. Carefully take the slide out of well 1 and dip it in the washing buffer in well 2. Incubate for 5 minutes while shaking on a rocking shaker.
  9. Repeat steps 7-8 twice by transferring the slide to well 3 and then to well 4.
  10. Decant all the liquid from well 4 and add 5 mL of water.
  11. Incubate for 2 minutes.
  12. Carefully remove the slide from the incubation tray and allow to air dry completely for at least 20 minutes (protected from light). Do not use a centrifuge to facilitate drying as this may cause a “comet” effect of the antibodies.
    Note: The slide should be absolutely dry before the scanning procedure. Water may cause background problems.

Materials

     
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