Journal: Database: The Journal of Biological Databases and Curation

Article Title: Phosprof: pathway analysis database of drug response based on phosphorylation activity measurements

doi: 10.1093/database/baac072

Figure Lengend Snippet: Scheme of the data collection and analysis for Phosprof. (Left) A drug from the Selleck L2400 Library was added to the cell culture media 2 h prior to the harvest of the cell lysate, which was then applied to a protein array with ATP. The tyrosine residues of the proteins on the array are phosphorylated by the cell lysate and detected using 4G10 antibody. (Right) The resultant ‘phosphorylation profiles’ were then analyzed to identify the significant pathways. The collected data can be browsed using various pathway analysis tools.

Article Snippet: Cell lysates including 100 μg of total protein were applied to an array with additional ATP at 30°C for 3 h. After the termination of the kinase reaction, the array was washed with Tris Buffered Saline with 0.05% Tween20 (TBST) and stained with the 4G10 phosphorylated protein-specific antibody (Merck, #05-1050) and a secondary fluorescein-conjugated antibody (Thermo, #A21235) to detect the phosphorylated tyrosine residues.

Techniques: Cell Culture, Protein Array