The expression levels of 308 mouse target proteins can be simultaneously detected, including cytokines, chemokines, adipokine, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules and other proteins, in cell culture supernatant, serum and plasma using the RayBio® Mouse Antibody Array. Furthermore, a biotin-label based internal control is used to normalize the protein levels in samples. The first step in using the RayBio Biotin label-based mouse antibody array 1 is to biotinylate the primary amine of the proteins in cell culture supernatants, serum or plasma. The biotin-labeled sample is then added onto the glass chip and incubated at room temperature. Fluorescent dye-conjugated streptavidin (Cy3 equivalent) is used to visualize the signals.
The relevant contents of the kit are as follows:
1. RayBio® Biotin label-based Mouse antibody array 1 Glass Chip with Frame
2. Dialysis tubes
3. Biotin-labeling reagent
4. Internal control
5. Stop solution
6. Blocking buffer
7. 20X wash buffer I
8. 20X wash buffer II
9. Fluorescent dye-conjugated streptavidin
10. Adhesive film
11. Serum buffer
Additional materials required are 1X PBS, pH 8.0, plate shaker, 1 ml tube, 15 ml and 50 ml conical collection tube, laser scanner for fluorescence detection and aluminum foil.
The cell culture supernatants, serum or plasma is dialyzed with a dialysis tube before the biotin-labeling procedure. We recommend loading 5-fold diluted serum or plasma with 1X PBS (pH 8) (80 ul serum or plasma + 320 ul 1X PBS) into a dialyzer and dialyzing with at least 500 ml 1X PBS buffer (pH = 8) at 4°C overnight. Briefly spin down the internal control tube before use. Add 40 ul of prepared internal control into each tube containing 400 ul dialyzed cell culture supernatants. Add the appropriate amount of prepared biotin labeling reagent into the above tube with sample and mix well immediately. Incubate the reaction solution at room temperature for 30 min with gentle shaking. Add 5 ul stop solution into the above reaction solution and immediately dialyze. Samples should be centrifuged at 10,000 g for 5 min (4°C) after dialysis. Air dry the chip for 1 hour in a clean environment before use. Add 400 ul of blocking buffer into each well and incubate at room temperature for 30 min to block slides. Decant blocking buffer from each well. Add 400 ul of each sample into the appropriate wells. Incubate arrays with sample at room temperature for 2 hours with gentle shaking or 4°C for overnight. Decant the samples from each well, and wash 3 times with 800 ul of 1X wash buffer I followed by wash buffer II at room temperature with gentle shaking for 5 min per wash. Add 400 ul of 1X fluorescent dye-conjugated streptavidin 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. Incubate at room temperature with gentle shaking for 2 hours. 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. Gently put the glass chip into a 50 ml centrifuge tube with 40 ml of 1X wash buffer I. Gently roll the tube or shake for 10 min. Remove the wash buffer. Repeat 2 times for a total of 3 washes. Wash the glass chip with 40 ml of 1X wash buffer II. Repeat one time for a total of two washes for 5 min per wash. Finally, wash the glass chip with 40 ml of deionized or distilled water for 5 min. Put the glass chip into a 50 ml centrifuge tube, and dry the glass chip by centrifuge at 1,000 rpm for 3 minutes. For the analysis, the signals can be visualized with a laser scanner, such as the Axon GenePix, using the Cy3 channel. A biotinylated protein and internal control will produce positive control signals, which can be used to identify the orientation and help normalize the results from different arrays being compared. If you prefer, the chip can be analyzed using services provided by RayBiotech Inc. technicians for an additional fee.
We have used the mouse protein antibody assay kit in our laboratory for identifying the differential expression of cytokines, growth factors, chemokines and receptor proteins in basement membrane extracts. The ease of detection, quantification and comparison of positive experimental samples to negative controls (i.e. basal media without exposure to cells) has been excellent in our experience. We truly recommend this assay kit for measurement of variety of proteins in murine cell culture media samples.