RESUMO
OBJECTIVE: To define the optimal conditions for preparing protein chips on aldehyde-coated slides. METHODS: The proteins were diluted in PBS containing 40% glycerol and spotted on aldehyde-coated slides. After the spots were dried for 1 hour at room temperature, the slides were stored at 4 degrees Celsius;. Following block and rinse, the slides were used for immunoassay and the results detected with a scanner. Several key factors that might influence the results were tested, including the number of spots, concentration of protein in the spotting solution, time of immobilization and the blocking reagent. RESULTS: Pre-spotting of 10 to 15 spots achieved good homogeneity of the subsequent spots on aldehyde-coated slides. The protein immobilized at 4 degrees Celsius; for 24 to 48 h showed higher fluorescence intensity and clearer images, and the slides blocked with 3% BSA produced the lowest background signal. The concentration of protein in the spotting solution significantly affected the fluorescence intensity. CONCLUSION: To ensure good results in preparing protein chips on aldehyde slides, pre-spotting of 10-15 spots can be necessary followed by immobilization at 4 degrees Celsius; for 24-48 h and 3% BSA blocking.
