Table 1.
Kinase-substrate pairs.
| Solution Assay | Protein Array Assay | |||
| Z_Score | ||||
| Upstream Kinases | Substrates | (Signal/Background) | -BSA | +BSA |
| CaMKII | Tau Protein | 16.01 | 15.00 | 10.1 |
| CDK5/p35 | Tau Protein | 28.24 | 18.80 | 6.5 |
| CK2 | pTEN | 67.65 | 12.80 | 18.6 |
| ERK2(MAPK) | 4EBP/PHAS-1 | 3.85 | 6.10 | 9.6 |
| ERK2(MAPK) | Elk-1 Fusion Protein | 8.17 | 17.00 | 20 |
| ERK2(MAPK) | Tau Protein | 3.34 | 24.10 | 19.4 |
| GSK3beta | GST-Axin | 33.63 | 1.40 | 2.4 |
| JNK1 | c-Jun 1–79 | 6.84 | 19.20 | 103.3 |
| MAP2K6(MKK6) | MAPK12 Inactive | 20.45 | 0.90 | 0.1 |
| MAP2K6(MKK6) | MAPK14, Inactive | 42.02 | 12.50 | 23.6 |
| MAPK14 (p38alpha) | 4EBP/PHAS-1 | 3.58 | 9.90 | 17.5 |
| MAPK14 (p38alpha) | ATF2 (aa 19–96) | 6.48 | 22.60 | 25.5 |
| MAPK14 (p38alpha) | MAPKAP-K2 Inactive | 13 | 13.90 | 19.4 |
| MAPK14 (p38alpha) | MAPKAP-K3 Inactive | 6.21 | 10.70 | 22.4 |
| MAPK14 (p38alpha) | MAPKAP-K5, Inactive | 5.07 | 5.40 | 2.4 |
| MKK4/SKK1 | MAPK12 Inactive | 17.43 | 1.80 | 7.7 |
| MKK4/SKK1 | MAPK8 (JNK1) Inactive | 23.17 | 2.90 | 0.7 |
| MKK4/SKK1 | MAPK9 (JNK2) Inactive | 6.61 | 0.90 | 5.9 |
| p38β2/SAPK2b2 | MAPKAP-K3 Inactive | 12.63 | 3.40 | 9.4 |
| PDK1 | AKT2 Inactive | 6.33 | 4.30 | 3.1 |
| PKA | ATF2 (aa 19–96) | 8.34 | 3.40 | 0.7 |
| PKA | Tau Protein | 12.66 | 33.90 | 4.6 |
| ROKα/ROCK-II | MYPT1 (654–880) | 51.54 | 14.40 | 23 |
| RPS6KA3(RSK2) | Estrogen Receptor-α | 9.08 | 0.40 | 2.7 |
A set of 24 kinase-substrate interactions comprising 14 different protein kinases and 18 substrates was defined as the test set for benchmarking protein microarray results against solution phase assays. Solution phase reactions were run for each of these pairs in the presence of 33P-ATP. After 1 hour, reactions were terminated by the addition of sample buffer and run on SDS-PAGE gels. Gels were subsequently dried, exposed to a phosphoscreen, and imaged in a Cyclone phosphoimager. Pixel intensity data from the resultant high resolution images was extracted and used to calculate the reported signal/background values. Protein microarray assays were run by incubating arrays with exogenous kinase in the presence of 33P-ATP. After 60 minutes, arrays were washed, dried, exposed to a phosphoscreen, and imaged using a Cyclone phosphoimager. Pixel intensity data from the resultant high resolution images was extracted using GenePix software, and used to calculate the reported Z-Scores.