A. Dialysis of Sample The saliva sample should be dialyzed with a Dialysis tube (Item A) before the biotin-labeling procedure. We recommend loading 200 μl saliva into a dialyzer and dialyzing with at least 500 ml 1X PBS buffer (pH= 8) at 4 0C. Change the 1X PBS buffer and dialyze again. Allow at least 3 h for each dialysis step, stir gently. The sample total volume may be changed after dialysis. B. Biotin-labeling Sample Avoid contamination with any solution containing amines (i.e., Tris, glycine) as well as Azide during the biotinylation process. 1. Briefly spin down Internal Control tube (Item C) before use. Add 100 μl 1X PBS, pH=8.0 into the Internal Control tube, pipette up and down to dissolve the powder. Add 40 μl prepared Internal Control into a new tube containing 35 μl dialyzed serum or plasma sample and 155 μl Serum Buffer (Item K). 2. Immediately before use, briefly spin down the Labeling Reagent tube (Item B). Add 100 μl 1X PBS into the tube, pipette up and down or vortex slightly to dissolve the powder to prepare 1X Labeling Reagent Solution. 3. Add an appropriate amount* of prepared Labeling Reagent into above tube with sample in step 2, mix well immediately. Incubate the reaction solution at room temperature with gentle shaking for 30 min. Gently tap the tube to mix the reaction solution every 5 min. * For labeling saliva: 36 μl of 1X Labeling Reagent Solution for labeling 1 mg total protein. 4. Add 3 μl Stop Solution (Item D) into above reaction solution and immediately dialyze as directed in Step B. 5. Samples should be centrifuged at 10,000 g for 5 min (4°C) after Dialysis. C. Dry the Glass Chip Open the box containing glass chip, and take it out, and then let it air dry for 1 hour in a clean environment before use. Note: Protect the chip from dust or others contaminants. D. Blocking and Incubation of Antibody Array 1. Add 400 μl of Blocking Buffer (Item F) into each well (Glass Chip with Frame, Item E) and incubate at room temperature for 1h to block slides. Make sure there are no bubbles are in the well. 2. Decant Blocking Buffer from each well (make sure to remove all of buffer). Add 400 μl of each sample (diluted 1:10 in blocking buffer) into appropriate wells. Incubate arrays with sample at room temperature for 2 hours with gentle shaking or 4 °C for overnight. 3. Decant the samples from each well, and wash 3 times with 800 μl of 1X Wash Buffer I (Item G) at room temperature with gentle shaking for 5 min per wash. 4. Put the glass chip with frame into a box with 1X Wash Buffer I (cover the whole glass slide and frame with Wash Buffer I), and wash 2 times at room temperature with gentle shaking for 10 min per wash. 5. Decant the Wash Buffer I from each well, Put the glass chip with frame into the box with Wash Buffer II (cover the whole glass slide and frame with Wash Buffer II), and wash 2 times at room temperature with gentle shaking for 5 min. 6. Remove all of Wash Buffer II in the well. Add 400 μl of 1X Fluorescent dye-Conjugated Streptavidin (cy3 equivalent) to each subarray. Cover the incubation chamber with Adhesive film, and then cover the plate with aluminum foil to avoid exposure to light or incubate in dark room (avoid array slide exposure to light in the following steps 7, 8, 9, 10 and 11). Note: briefly spin down the Fluorescent dye-Conjugated Streptavidin (cy3 equivalent) (Item I) before use. Add 1000 μl of Blocking Buffer into the tube to prepare a Streptavidin Concentrate. Pipette up and down to mix gently (don’t store the Concentrate for next day use). Add 200 μl of Streptavidin Concentrate into a tube with 800 μl of Blocking Buffer. Mix gently to prepare 1 X Streptavidin solution. 7. Incubate at room temperature for 2 hours with gentle shaking 8. Decant the solution and disassemble the slide out of the incubation frame and chamber. Disassemble the device by pushing clips outward from the slide side. Carefully remove the slide from the gasket. 9. Gently put the glass chip into a 30 ml centrifuge tube provided with 30 ml of 1X Wash Buffer I (add enough Wash Buffer I to cover the whole slide). Gently shake for 10 min. Remove the wash buffer. Repeat 2 times for a total of 3 washes. 10. Wash the glass chip with 30 ml of 1X Wash Buffer II. Repeat one time for a total of two washes for 5 min per wash. 11. Finally wash the glass chip with 30 ml of deionized or distilled water for 5 min. E. Fluorescence Detection Put the glass chip into a 30 ml centrifuge tube provided, and dry the glass chip by centrifuge at 1,000 rpm for 3 minutes. You can also let the glass chip dry completely in a clean environment (protect from light). Make sure the slides are absolutely dry before the scanning procedure. The signals can be visualized with a laser scanner, such as the Axon GenePix, using the cy3 channel.
