Unbiased Screening of Activated Receptor Tyrosine Kinases (RTKs) in Tumor Extracts Using a Mouse Phospho-RTK Array Kit

Materials and Reagents

Common lab consumables

1. Pipette tips 
2. Gloves 
3. Plastic containers with the capacity to hold 50 ml (for washing the arrays)
4. Plastic wrap
5. Absorbent lab wipes (KimWipes^® or equivalent) 
6. Paper towels
7. Plastic transparent sheet protector (trimmed to 10 cm x 12 cm and open on three sides) 
8. TissueRuptor Disposable Probes (QIAGEN, catalog number: 990890)

1. Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, catalog number: ARY014)
   Kit Contents:
   1. 4 Array Membranes
   2. 4-Well Multi-dish
   3. Array Buffers:
      1)

      Lysis Buffer 17 (see Recipes)
      2)

      Array Buffer 1
      3)

      1x Array Buffer 2 (see Recipes)
      4)

      1x Wash Buffer (see Recipes)
      5)

      Chemi Reagent Mix (see Recipes)
   4. Lysis Buffer
   5. Wash Buffer
   6. Anti-Phospho-Tyrosine-HRP Detection Antibody
   7. Chemiluminescent Detection Reagents
   8. Transparency Overlay Template
   9. Detailed Protocol
   Note: Store the unopened kit at 2 °C to 8 °C. Do not use past kit expiration date.
2. Aprotinin (Sigma, catalog number: A6279)
3. Leupeptin (Tocris, catalog number: 1167)
4. Pepstatin (Tocris, catalog number: 1190)

1. PDVF membranes
2. CL-XPosure^TM Film, 5 x 7 in. (13 x 18 cm) (Thermo Fisher Scientific, catalog number: 34090)
3. 4x Laemmli Sample Buffer (Bio-Rad Laboratories, catalog number: 1610747)
4. 4%-20% Mini-PROTEAN^® TGX Stain-Free^TM Protein Gels, 12-well, 20 µl (Bio-Rad Laboratories, catalog number: 4568095)
5. 10x Tris/Glycine Buffer for Western Blots and Native Gels (Bio-Rad Laboratories, catalog number: 1610771)
6. 10x Tris/Glycine/SDS (Bio-Rad Laboratories, catalog number: 1610772)
7. Methanol ACS reagent, ≥ 99.8% (Sigma, catalog number: 179337)
8. Pierce^TM BCA Protein Assay Kit (Thermo Scientific, catalog number: 23227)
9. Blotting-Grade Blocker, nonfat dry milk (Bio-Rad Laboratories, catalog number: 1706404)
10. Tween 20, 100% Nonionic Detergent (Bio-Rad Laboratories, catalog number: 1706531)
11. TBS Buffer, 20x liquid (VWR, catalog number: 97064-338)
12. Restore^TM PLUS Western Blot Stripping Buffer (Thermo Scientific, catalog number: 46430)
13. SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, catalog number: 34577)
14. Primary Antibodies:
    cKIT and p-cKIT (Cell Signaling Technology, catalog number: 9370)
    PDGFRA and p-PDGFRA (R&D Systems, catalog numbers: AF307 and AF2114)
15. HRP-labeled secondary antibodies (Promega, catalog number: W4011)
16. Deionized or distilled water
17. 1x Tris/Glycine running buffer (see Recipes)
18. Transfer buffer (see Recipes)
19. 1x TBS (1 L) (see Recipes)
20. TBS/Tween (see Recipes)
21. 5% Blotting-Grade Blocker, nonfat dry milk/TBS/Tween (see Recipes)

Equipment

1. Pipettes (Gilson, Pipetman Classic)
2. Flat-tipped tweezers
3. Autoradiography cassette
4. Rocking platform shaker
5. TissueRuptor II (QIAGEN, catalog number: 9002755)
6. Centrifuge 5810R (Eppendorf, catalog number: 5811000010)
7. Film developer Alphatek AX-390-SE (Alphatek)
8. Rocking platform shaker
   

Software

1. Protein Array Analyzer for ImageJ (Author: Gilles Carpentier, Faculte des Sciences et Technologies, Universite Paris Est Creteil Val de Marne, France, https://imagej.nih.gov/ij/macros/toolsets/Protein%20Array%20Analyzer.txt)
2. GraphPad Prism 7.0a (GraphPad Software, Inc., www.graphpad.com)
   

Procedure

1. Reagent Preparation (Mouse Phospho-RTK Array Kit)
   Note: Bring all reagents to room temperature before use.
   1. Mouse Phospho-RTK Array
      Four nitrocellulose membranes each containing 39 different anti-RTK antibodies printed in duplicate. Handle arrays only with gloved hands and flat-tipped tweezers. 
   2. Anti-Phospho-Tyrosine-HRP Detection Antibody: 50 μl of mouse anti-phospho-tyrosine antibody conjugated to HRP
      Immediately before use, dilute the Detection Antibody to the working concentration specified on the vial label using 1x Array Buffer 2.
      Note: Prepare only as much Detection Antibody as needed to run each experiment.
   3. Lysis Buffer 17 (see Recipe 5)
   4. 1x Array Buffer 2 (see Recipe 6)
   5. 1x Wash Buffer (see Recipe 7)
   6. Chemi Reagent Mix (see Recipe 8)
      
      
2. Sample Preparation
   1. Excise by dissection and disrupt 1-5 mg of tumor tissue (approx 0.2-0.3 cm³) using TissueRuptor II in 1 ml Lysis Buffer 17 prepared with protease inhibitors. 
   2. Centrifuge samples at 10,621 x g for 10 min at 4 °C in an Eppendorf Centrifuge 5810R. 
   3. Collect supernatant and quantified protein concentrations in tumor lysates using the Pierce^TM BCA Protein Assay Kit.
   Notes:
   1. The suggested starting range for tissue lysates is 100-300 μg and the maximum allowable lysate volume is 250 μl/array. 
   2. Tissue lysates should be used immediately or aliquoted and stored at ≤ -70 °C.
   3. Thawed lysates should be kept on ice prior to use.
      
      
3. Array Procedure
   Note: Bring all reagents to room temperature before use. Keep samples on ice. To avoid contamination, wear gloves while performing the procedures.
   1. Prepare all reagents and samples as directed in the previous sections. 
   2. Pipette 2.0 ml of Array Buffer 1 into each well of the 4-well Multi-dish that will be used. Array Buffer 1 is used as a block buffer. 
   3. Using flat-tip tweezers, remove each array to be used from between the protective sheets. 
   4. Place one array into each well of the 4-well Multi-dish. The array number should be facing upward.
      Note: Upon contact with Array Buffer 1 the blue dye will disappear from the spots. The capture antibodies are retained in their specific locations. 
   5. Incubate for 1 h at room temperature on a rocking platform shaker. Orient the tray so that each array rocks from end to end in its well. 
   6. While the arrays are blocking, prepare samples by diluting the desired quantity of lysate in 1.25 ml of Array Buffer 1. Adjust to a final volume of 1.5 ml with Lysis Buffer 17 as necessary. The maximum allowable lysate volume is 250 μl/array. 
   7. Aspirate Array Buffer 1 from the 4-well Multi-dish. Add the prepared samples and place the lid on the 4-well Multi-dish. 
   8. Incubate overnight at 2-8 °C on a rocking platform shaker.
      Note: A shorter incubation time may be used if optimal sensitivity is not required. 
   9. Carefully remove each array and place into individual plastic containers with 20 ml of 1x Wash Buffer. Rinse the 4-well Multi-dish with deionized or distilled water and dry thoroughly. 
   10. Wash each array with 1x Wash Buffer for 10 min on a rocking platform shaker. Repeat two times for a total of three washes. 
   11. Dilute the Anti-Phospho-Tyrosine-HRP Detection Antibody in 1x Array Buffer 2 using the dilution factor on the vial label. Pipette 2.0 ml into each well of the 4-well Multi-dish. 
   12. Carefully remove each array from its wash container. Allow excess buffer to drain from the array. Return the array to the 4-well Multi-dish containing the Anti-Phospho-Tyrosine-HRP and cover with the lid. 
   13. Incubate for 2 h at room temperature on a rocking platform shaker. 
   14. Wash each array as described in Steps C9 and C10.
       Note: Complete the remaining steps without interruption. 
   15. Carefully remove each membrane from its wash container. Allow excess Wash Buffer to drain from the membrane by blotting the lower edge onto paper towels. Place each membrane on the bottom sheet of the plastic sheet protector with the identification number facing up. 
   16. Pipette 1 ml of the prepared Chemi Reagent Mix evenly onto each membrane.
       Note: Using less than 1 ml of Chemi Reagent Mix per membrane may result in incomplete membrane coverage. 
   17. Carefully cover with the top sheet of the plastic sheet protector. Gently smooth out any air bubbles and ensure Chemi Reagent Mix is spread evenly to all corners of each membrane. Incubate for 1 min. 
   18. Position paper towels on the top and sides of the plastic sheet protector containing the membranes and carefully squeeze out excess Chemi Reagent Mix. 
   19. Remove the top plastic sheet protector and carefully lay an absorbent lab wipe on top of the membranes to blot off any remaining Chemi Reagent Mix. 
   20. Leaving membranes on the bottom plastic sheet protector, cover the membranes with plastic wrap taking care to gently smooth out any air bubbles. Wrap the excess plastic wrap around the back of the sheet protector so that the membranes and sheet protector are completely wrapped. 
   21. Place the membranes with the identification numbers facing up in an autoradiography film cassette.
       Note: Use an autoradiography cassette that is not used with radioactive isotope detection.
   22. Expose membranes CL-XPosure^TM Film. Multiple exposure times are recommended (Figure 1).
       
       
       Figure 1. Proteomic analysis of Receptor tyrosine kinases in mouse KSHV-induced KS-like tumors shows activation of PDGF receptor-alpha. Mouse Phospho-Receptor Tyrosine Kinase (RTK) Array Kit used to quantify levels of phosphorylation of 39 RTKs in mouse KSHV-induced KS-like tumors. Note the major activation spot corresponding to PDGF receptor alpha chain (Cavallin et al., 2018).

Data analysis

The positive signals seen on developed film can be quickly identified by placing the transparency overlay template on the array image and aligning it with the pairs of reference spots in three corners of each array. The stamped identification number on the array should be placed on the left-hand side. The location of controls and capture antibodies is listed in the Appendix.

Notes:

1. Reference spots are included to align the transparency overlay template and to demonstrate that the array has been incubated with Anti-Phospho-Tyrosine-HRP during the assay procedure. 
2. Pixel densities on developed X-ray film can be collected and analyzed using a transmission mode scanner and image analysis software.

1. Using ImageJ Protein Array Analyzer tool create a template to analyze pixel density in each spot of the array. 
2. Export signal values to a spreadsheet file for manipulation in a program such as Microsoft Excel or GraphPad Prism. 
3. Determine the average signal (pixel density) of the pair of duplicate spots representing each RTK. 
4. Subtract an averaged background signal from each RTK. Use a signal from a clear area of the array or the PBS negative control spots as a background value (Figure 2).
   
   
   Figure 2. Quantification of the 45 min exposure array from Figure 1. Bar graph and pie chart from densitometry for the higher-exposure blot are equally color coded for the most prominent signals (Cavallin et al., 2018).

Western Blotting
Twenty micrograms of proteins of the same tumor lysates used for the Mouse Phospho-RTK Array were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE in 4-20% Mini-PROTEAN^® TGX Stain-Free^TM Protein Gels, 12-well. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane by blotting overnight at 300 mA. Membranes were blocked with 5% nonfat milk/TBS/Tween 20 for 1 h and incubated with primary antibodies (4 °C, 16 h). After 3 TBS/Tween 20 washes, membranes were incubated with HRP-labeled secondary antibodies (1:10,000 dilution) for 1 h at room temperature. Protein bands were developed using SuperSignal^TM West Pico PLUS Chemiluminescent Substrate. To analyze multiple proteins on the same membrane, membranes were washed with Restore PLUS Western Blot Stripping Buffer according to the manufacturer’s protocol (Figure 3).


Figure 3. Validation of the mouse Phospho-receptor Tyrosine Kinase (RTK) array kit. PDGFRA and phospho-PDGFRA (A) or c-KIT and phospho-cKIT (B) determined in 3 different samples of Mouse Normal Skin and mouse KSHV-induced KS-like tumors from 3 different mice by immunoblotting (Cavallin et al., 2018). (C) GAPDH used as loading control.

Recipes

1. 1x Tris/Glycine running buffer (1 L)
   100 ml 10x Tris/Glycine
   900 ml distilled water
2. Transfer buffer (1 L)
   100 ml of 10x Tris/Glycine/SDS
   200 ml Methanol
   700 ml distilled water
3. 1x TBS (1 L)
   50 ml 20x TBS
   950 ml distilled water
4. TBS/Tween (500 ml)
   25 ml TBS 20x
   1 ml Tween 20
   Distilled water to 500 ml
5. 5% Blotting-Grade Blocker, nonfat dry milk/TBS/Tween (10 ml)
   500 mg Blotting-Grade Blocker, nonfat dry milk
   10 ml TBS/Tween
   

Note: Bring all reagents to room temperature before use.

6. Lysis Buffer 17
   Add 10 μg/ml Aprotinin, 10 μg/ml Leupeptin, and 10 μg/ml Pepstatin to the volume of Lysis Buffer 17 required for cell lysate preparation
   Prepare fresh for each use
7. 1x Array Buffer 2
   Add 2 ml of Array Buffer 2 Concentrate to 8 ml of deionized or distilled water
   Prepare fresh for each use
   
8. 1x Wash Buffer
   If crystals have formed in the concentrate, warm the bottles to room temperature and mix gently until the crystals have completely dissolved
   Dilute 40 ml of 25x Wash Buffer Concentrate into 960 ml of deionized or distilled water
9. Chemi Reagent Mix
   Chemi Reagents 1 and 2 should be mixed in equal volumes within 15 min of use (protect from light)
   1 ml of the resultant mixture is required per membrane
   

Acknowledgments

This work was supported by NIH grants CA75918, and CA136387; and by NCI/OHAM supplements from the Miami CFAR grant 5P30AI07396, and by the Florida Biomedical Foundation, Bankhead Coley Foundation Grant 3BB05.

Competing interests

The authors do not have any conflicts of interests or competing interests.

Ethics

The animal experiments have been performed under UM IACUC approval number 13±093. The University of Miami has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. Additionally, UM is registered with USDA APHIS. The Council on Accreditation of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) has continued the University of Miami's full accreditation.

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