人 RTK Phosphorylation Array G1

• High sensitivity and specificity
• Low sample volume (10-100 μL per array)
• Large dynamic range of detection
• Compatible with most sample types
• Test 4 or 8 samples on each slide
• Suitable for high-throughput assays

The G-Series arrays feature fluorescent signal detection. The antibodies are spotted on glass slide solid supports and require a laser scanner for data collection.
All G-Series arrays work on the sandwich ELISA principle, utilizing a matched pair of antibodies: an immobilized capture antibody and a corresponding biotinylated detection antibody.

1. Dry the glass slide
2. Block array surface
3. Incubate with Sample
4. Incubate with Biotinylated Detection Antibody Cocktail
5. Incubate with Streptavidin-Conjugated Fluor
6. Disassemble the glass slide
7. Scan with a gene microarray laser scanner
8. Perform densitometry and analysis

1. Protease Inhibitor Cocktail: Briefly spin down the Protease Inhibitor Cocktail tube before use. Add 60 µl of 1x Lysis Buffer into the vial to prepare a 100X Protease Inhibitor Cocktail. 2. Phosphatase Inhibitor Cocktail Set II: Briefly spin down the Phosphatase Inhibitor Cocktail Set II tube before use. Add 180 µl of 1X Lysis Buffer into the vial to prepare 25X Phosphatase Inhibitor Cocktail Set II Concentrate . Dissolve the powder thoroughly by a gentle mix. 3. 2X Cell Lysis Buffer: Cell lysis buffer should be diluted 2-fold with deionized or distilled water. Add 20 µl of Protease Inhibitor Cocktail Concentrate and 80 µl of Phosphatase Inhibitor Cocktail Set II Concentrate into 1.9 ml of 1X Lysis Buffer before use. Mix well. 4. 20X Washing Buffer I or II : If the 20X Wash Concentrate contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 25 ml of Wash Buffer Concentrate into deionized or distilled water to yield 500 ml of 1X Wash Buffer. 5. Biotinylated anti-Phosphotyrosine: Briefly spin the Detection Antibody tube before use. Add 65 µl of Blocking Buffer into the tube to prepare a Biotinylated Anti-phosphotyrosine Concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). Add 30 µl of Detection Antibody Concentrate into a tube with 570 µl of Blocking Buffer. Mix gently to prepare 1X Biotinylated Anti-phosphotyrosine. 6. Fluorescent dye-Conjugated Streptavidin (cy3 equivalent): briefly spin the Fluorescent dye-Conjugated Streptavidin before use. Add 50 µl of Blocking Buffer into the tube to prepare a Streptavidin Concentrate. Pipette up and down to mix gently. Add 10 µl of Streptavidin Concentrate into a tube with 1 ml of Blocking Buffer. Mix gently to prepare 1X Streptavidin solution.

Use serum-free conditioned media if possible. If serum-containing conditioned media is required, it is highly recommended that complete medium be used as a control since many types of sera contains cytokines. We recommend the following parameters for your samples: 50 to 100 µl of original or diluted serum, plasma, cell culture media, or other body fluid, or 50-500 µg/ml of protein for cell and tissue lysates. If you experience high background or if the fluorescent signal intensities exceed the detection range, further dilution of your sample is recommended.

Take out the glass slide from the box, and let it equilibrate to room temperature inside the sealed plastic bag for 20-30 minutes. Remove slide from the plastic bag, peel off the cover film, and let it air dry for another 1-2 hours.

Blocking & Incubation
1. Add 100 µl Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides.
2. Decant buffer from each well. Add 100 µl of sample to each well. Incubate arrays at room temperature for 1-2 hour.
3. Decant the samples from each well, and wash 5 times (5 min each) with 150 µl of 1X Wash Buffer I at room temperature with gentle shaking. Completely remove wash buffer in each wash step. Dilute 20x Wash Buffer I with H2O.
4. Decant the 1x Wash Buffer I from each well, wash 2 times (5 min each) with 150 µl of 1X Wash Buffer II at room temperature with gentle shaking.Completely remove wash buffer in each wash step. Dilute 20X Wash Buffer II with H2O.

Incubation with Biotinylated Antibody Cocktail & Wash
5. Reconstitute the detection antibody by adding 1.4 ml of Sample Diluent to the tube. Spin briefly.
6. Add 80 µl of the detection antibody cocktail to each well. Incubate at room temperature for 1-2 hour.
7. Decant the samples from each well, and wash 5 times (5 mins each) with 150 µl of 1X Wash Buffer I and then 2 times with 150 µl of 1x Wash Buffer II at room temperature with gentle shaking. Completely remove wash buffer in each wash step.

Incubation with Cy3 Equivalent Dye-Streptavidin & Wash
8. After briefly spinning down, add 1.4 ml of Sample Diluent to Cy3 equivalent dye-conjugated streptavidin tube. Mix gently.
9. Add 80 µl of Cy3 equivalent dye-conjugated streptavidin to each well. Cover the device with aluminum foil to avoid exposure to light or incubate in dark room. Incubate at room temperature for 1 hour.
10. Decant the samples from each well, and wash 5 times (5 mins each) with 150 µl of 1X Wash Buffer I at room temperature with gentle shaking. Completely remove wash buffer in each wash step.

Fluorescence Detection
11. Disassemble the device by pushing clips outward from the slide side. Carefully remove the slide from the gasket.
12. Place the slide in the Slide Washer/Dryer (a 4-slide holder/centrifuge tube), add enough 1x Wash Buffer I (about 30 ml) to cover the whole slide, and then gently shake at room temperature for 15 minutes. Decant Wash Buffer I. Wash with 1x Wash Buffer II (about 30 ml) and gently shake at room temperature for 5 minutes.
13. Remove water droplets completely by gently applying suction with a pipette to remove water droplets. Do not touch the array, only the sides.
14. Imaging: The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength (green channel) such as Axon GenePix.

Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software (GenePix, ScanArray Express, ArrayVision, MicroVigene, etc.).