The cells were centrifuged for 10 minutes at 10,000 rpm at 4 C. a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase specific substrates into macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 l of a kinase reaction solution containing cancer cell lysate and measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel with potential to measure the activity of as many as 5 kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752. BL21 strains containing the pGEX-4T1 vector with inserted amino acid sequences for Gab1 residues 431 to 561, Crkl residues 120 to 303, or EGFR pathway substrate 15 (Eps15) residues 758 to 881 were used to produce the fusion proteins GST-Gab1, GST-Crkl, and GST-Eps15 [12, 14, 22]. An additional BL21 strain containing the pGEX-4T1 vector with the inserted sequence for tensin residues SKLB1002 1392 to 1672 was used to produce GST-tensin. To produce these proteins, BL21 cells were grown in 2YT medium (16 g tryptone, 10 g yeast extract, 5 g NaCl in 1 L H2O) to an OD600 of 0.6. Protein production was induced using 1 mM isopropyl–d-thiogalactopyranoside for 4 hours at 37 C. Cells were centrifuged for 20 minutes. The supernatant was removed, and cells were washed with cold PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.7 mM KH2PO4) and centrifuged as before. The supernatant was again removed and BPER II Bacterial Protein Extraction Reagent with cOmplete Protease Inhibitor Cocktail was used according to manufacturers instructions to lyse the cells. To avoid clogging the purification column, the viscosity of the solution was reduced by mild sonication. The sample was sonicated for 15 Rabbit Polyclonal to BCLAF1 seconds, followed by 45 seconds rest, and the sonication procedure was repeated 4 additional times. The lysate was passed through an 18 gauge syringe needle and centrifuged for 20 minutes at 3720 g, and the resulting supernatant was recovered. The viscosity of the solution was further reduced by passing through a 25 gauge syringe needle and centrifuging a final time. The substrates were purified with a GST affinity column according to the manufacturers instructions and concentrated using a 30 kDa molecular weight cut off filter. The protein concentration was determined using a BCA assay and the purified protein was then aliquoted and stored at ?80 C until needed. Cell culture NCI-H1975 (H1975) lung adenocarcinoma cells, IMR-90 lung fibroblast cells, K562 CML cells, and HL60 acute myeloid leukemia cells were obtained from the American Type Culture Collection. H1975, K562, and HL60 cells were grown in RPMI-1640 medium supplemented with 300 mg/L glutamine and 10% fetal bovine serum as well as 100 units/ml penicillin and 100 g/ml streptomycin. IMR-90 cells were grown in MEM medium with 10% fetal bovine serum. For passaging and harvesting the adherent cultures, H1975 and IMR-90, cells were detached from the flask using trypsin-EDTA (0.25% trypsin, 1 mM EDTA). To harvest all cultures, the cells were removed from the flask and centrifuged to form a pellet. They were then resuspended and incubated for 20 minutes in mammalian cell SKLB1002 lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, cOmplete Protease Inhibitor Cocktail and 1 mM PMSF. The cells were centrifuged for 10 minutes at 10,000 rpm at 4 C. The supernatant was removed and stored at ?80 C until use, and the final protein concentration SKLB1002 was determined using a BCA assay. solution-phase kinase assay Solution phase kinase assays were performed by incubating 0.2 g/l GST-Gab1 or GST-Crkl, 0.2 g/l cell lysate, and 0.2 mM ATP in 1 kinase reaction buffer for 1 hour at 37 C. Kinase reaction buffer contains 50 mM.